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Surface glycosylation profiles of urine extracellular vesicles.

ABSTRACT: Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

SUBMITTER: Gerlach JQ

PROVIDER: S-EPMC3777961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Opiate abuse increases the risk of HIV transmission and exacerbates HIV neuropathology by increasing inflammation and modulating immune cell function. Exosomal EVs(xEV) contain miRNAs that may be differentially expressed due to HIV infection or opiate abuse. Here we develop a preliminary exosomal-miRNA biomarker profile of HIV-infected PBMCs in the context of opiate use. PBMCs infected with HIV were treated with increasing dosages of morphine for 72 hours, the culture supernatants were collected, and the exosomes isolated using differential centrifugation. Exosomal miRNAs were extracted, expression levels determined via Nanostring multiplexed microRNA arrays, and analyzed with Webgestalt. The effect of the exosomes on neuronal function was determined by measuring calcium. Preliminary findings show that HIV-1 infection altered the miRNA profile of PBMC-derived EVs concurrently with opiate exposure. MicroRNA, hsa-miR-1246 was up-regulated 12-fold in the presence of morphine, relative to uninfected control. PBMCs infected with HIV-1 MN, an X4-tropic HIV-1 strain and exposed to morphine, displayed a trend which suggests potential synergistic effects between HIV-1 infection and morphine exposure promoting an increase in viral replication. Dose-dependent differences were observed in miRNA expression as a result of opiate exposure. The xEVs derived from PBMCs exposed to morphine or HIV modulated neuronal cell function. SH-SY5Y cells, treated with xEVs derived from ART-treated PBMCs, exhibited increased viability while for SH-SY5Ys exposed to xEVs derived from HIV-1 infected PBMCs viability was decreased compared to the untreated control. Exposing SH-SY5Y to xEVs derived from HIV-infected PBMCs resulted in significant decrease in calcium signaling, relative to treatment with xEVs derived from uninfected PBMCs. Overall, HIV-1 and morphine induced differential miRNA expression in PBMC-derived exosomes, potentially identifying mechanisms of action or novel therapeutic targets involved in opiate use disorder, HIV neuropathology, TNF signaling pathway, NF-κB signaling pathway, autophagy, and apoptosis in context of HIV infection.

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Surface glycosylation profiles of urine extracellular vesicles.

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