Fig 1.
Isolation of exosomes: Schematic representation for the isolation procedure of exosomes from cell-conditioned media.
Fig 2.
Nucleotide sequence alignment of multiple variants of hminpp1: Three variants of the hminpp1 gene are identified.
These variants are formed by alternative splicing of the gene. Homology modeling of the full-length sequences of the three variants collected from the NCBI database were aligned to evaluate sequence similarity. hminpp1 variant1 (NM_004897.5) is the full-length gene (MINPP1-1). hminpp1 variant-2 (MINPP1-2) comprises similar 5’ sequence but lacks substantial 3’ sequence. hminpp1 variant-3 (MINPP1-3) lacks 5’ sequence but retains 3’ sequence.
Fig 3.
Minpp1 isoform-2 amino acid sequence analysis.
A). Multiple AAs sequence alignment of isoforms of hMinpp1. Three isoforms of hMinpp1 were identified as a translated outcome of the alternatively spliced hminpp1 gene. Homology modeling of the entire AAs sequences of three isoforms collected from NCBI sequence aligned to evaluate sequence similarity. AAs 213–278 were relatively conserved for all isoforms of hMinpp1. Isoform-1 (NP_004888.2) is a full-length protein. Isoform-2 (NP_001171588.1) comprises similar N-terminal sequences but lacks C-terminal sequences. Isoform-3 (NP_001171589.1) lacks the majority of N-terminal sequences but retains C-terminal sequences. The "NATA": N-Glycosylation site is conserved in all three isoforms, while glucose 1-phosphatase (DIDD) and ER retention (SDEL) motifs were absent in isoform-2, B). The InterPro protein viewer for the Minpp1-isoform-2 (NP_001171588.1). The underlying tool—InterProScan—annotates protein’s domain and motifs using predictive models provided by multiple databases [60], C). Summary of the prospective motifs in hMinpp1 isoform-2 based on its sequence alignment with hMinpp1 isoform-1.
Fig 4.
hMinpp1 isoform-2 (NP_001171588.1) (highlighted yellow) related amino acid sequences across species A). A rooted slanted cladogram B). BLASTP was used to compute the hMinpp1 isoform-2 amino acid sequence in a pair-wise alignment against the curated NCBI refseq_protein database to construct the above-mentioned distance-based cladogram. The higher pairing sequences were included in the tree, displaying homology across taxonomic species—percent identity ranged: 25.79% to 100%. The search was limited to records that exclude models (XM/XP). hMinpp1 isoform-1: NP_004888.2. hMinpp1 isoform-3: NP_001171589.1.
Fig 5.
Expression profile of hminpp1 transcript variants by real-time PCR in A). MCF-7 breast cancer cells. B). MCF-10A normal breast cells. C). a comparative expression analysis of hminpp1 variants. hminpp1 variants expression is normalized to 100% in MCF-7 cells to better analyze each variant’s difference compared to MCF-10A cells. D). hminpp1 variant1 expression in MCF-7 cells under different stress conditions. E). hminpp1 variant2 expression in MCF-7 cells under different stress conditions, (p<0.05) F). Comparative expression analysis of hminpp1 variant-1 and -2 under cellular stress. hminpp1 variant-1 expression is normalized to 100% in the control sample to analyze the relative difference in each variant better. Data, except in C, were converted into a percentage of control for better representation. Hydrogen peroxide (H2O2): 100μM; BFA: 10.0μg/mL. Data reported as mean values of mRNA abundance after normalization with 18S gene expression. The error bars represent the standard error of the mean (±SEM). One-way ANOVA with post-hoc Tukey HSD (Honestly Significant Difference) was used to measure the significance between groups with significance p<0.01 (A, B, C&, D) and p<0.05(E).
Fig 6.
Plasmid Expression analysis: Bright field and fluorescence images of MCF-7 breast cancer cells successfully transfected and incubated 24h with A). Minpp1 isoform-2 plasmid (GFP tagged), B). GFP only plasmid. C). Positively transfected cells were lysed, and SDS-PAGE resolved proteins were transferred onto nitrocellulose membrane to further immuno-stain with anti-Minpp1 polyclonal antibody (upper blot) and anti-GFP monoclonal antibody (lower blot). Results show that the polyclonal antibody raised against Minpp1 isoform-1 also shared its epitope with Minpp1 isoform-2. GFP MW: 27kDa. Lane-1, 5 & 6: Control cell lysate (CL), Lane-2 & 3: Minpp1 isoform-2 GFP CL, Lane-4: GFP alone CL.
Fig 7.
Confocal immunofluorescence image analysis on 4% paraformaldehyde (PFA) fixed MCF-7 cells: A). hMinpp1 isoform-2 GFP transfected MCF-7 cells were immuno-stained with Mouse anti-CD63 antibody (MVB/exosomes biomarker). B). hMinpp1 isoform-2 GFP transfected MCF-7 cells were immunostained with mouse anti-LAMP2 antibody (Lysosomal biomarker). C). Cells were immunostained with Rabbit anti-Minpp1 and Mouse anti-CD63 antibody (MVB/exosomes biomarker). D). Minpp1 isoform-1 OFPSpark and Minpp1 isoform-2 GFP transfected MCF-7 cells. E&F). Minpp1 isoform-1 OFPSpark and Minpp1 isoform-2 GFP transfected MCF-7 cells were stained with mouse anti-GRP-78 antibody (ER biomarker). Secondary antibody staining was done by goat anti-mouse Alexa Fluor 555 (Red); Goat anti-mouse Alexa flour 488 (Green); Goat anti-mouse Alexa Fluor 647 (far Red); and goat anti-rabbit Alexa flour 555 (Red). Nuclei were stained with DAPI (blue). Scale bar is 10μm.
Fig 8.
Characterization of exosomes: TEM micrographs of heterogeneous exosomes isolated from A). Control MCF-7 cells, and B). MCF-7 cells treated with BFA (10μg/mL) inhibitor for 24hr. Scale bar: 200nm C). Nanoparticle tracking analysis of isolated exosomes. The distribution peaks around ~120 nm, following the exosome enrichment during preparation. D). An uninterrupted secretion of smaller exosomes, distribution peaks around ~95 nm, were reported in the presence of a BFA (10μg/mL) inhibitor for 24hr. 1% Uranyl Acetate for 15sec at RT was used to stain the samples negatively.
Fig 9.
Analysis of Minpp1 isoform-2 expression in BFA induced MCF-7 exosomes: A). Mechanistic illustration of protein trafficking inhibitor-BFA, B). Western blot analysis of exosomes (Exo) isolated from BFA (10μg/mL, 24h) treated and untreated MCF-7 cells conditioned media, pre-enriched by sequential centrifugation. An equal amount of proteins (7μg) from both control and BFA-treated Exo were loaded. Probing was done by; anti-Minpp1 antibody (Fabgennix Inc.) that also binds to Minpp1 isoform-2, anti-Exo biomarker: CD63 antibody (Santa Cruz Biotech Inc.) and, β-Actin antibody (Santa Cruz Biotech Inc.) at 1:1000 dilution in blocking buffer, CL; cell lysate (40μg protein). Shown blots are a representative result of three independent experiments (n = 3). C). A comparative analysis of Minpp1’s expression percentage) between BFA treated and untreated Exo sample. A more than 3-fold increase in Minpp1 isoform-2 secretion in EVs from BFA treated cells was observed.
Fig 10.
Enzymatic analysis of exosome-based Minpp1 isoform-2.
A). Competitive inhibition enzyme immunoassay against exosomes pre-enriched by ultra-centrifugation. The ELISA plate was pre-coated with goat anti-rabbit antibody. Samples were subjected to the plate along with antibodies specific to InsP3 and Horseradish Peroxidase (HRP)-conjugated InsP3. The competitive inhibition reaction is launched between HRP labeled InsP3 and unlabeled InsP3 with the antibody. On adding HRP substrate solution, color develops reciprocally to the amount of InsP3 in the sample. Samples were spiked with 5μM InsP4 for 24h RT. Exosomes (~1.0μg) were resuspended in Minpp1 assay Buffer with 16mM CHAPS. Microsomes (~1.0μg) were resuspended in Minpp1 assay Buffer with 3mM CHAPS. The bar graph shows the percentage of InsP4 dephosphorylation as a qualitative activity of Minpp1 isoform-2 enzyme compared to control (InsP4+Minpp1 Assay Buffer). B). Hydrolysis of InsP6 (4 nmol) with exosomes as an indication of Minpp1 isoform-2 enzyme activity. Freshly collected serum-free conditioned media was differentially ultra-centrifuged to isolate exosomes. The isolated exosomes were resuspended in Minpp1 enzymatic assay buffer and later incubated overnight with InsP6 (4 nmol) at room temperature. After incubation, metabolized InsPs were resolved by PAGE and visualized with toluidine blue staining.
Fig 11.
Pictorial depiction of protein secretory pathway: Conventional vs. Unconventional protein pathway.