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Surface glycosylation profiles of urine extracellular vesicles.

TitleSurface glycosylation profiles of urine extracellular vesicles.
Publication TypeJournal Article
Year of Publication2013
AuthorsGerlach JQ, Krüger A, Gallogly S, Hanley SA, Hogan MC, Ward CJ, Joshi L, Griffin MD
JournalPLoS One
Volume8
Issue9
Paginatione74801
Date Published2013
ISSN1932-6203
KeywordsAdult, Carbohydrates, Cell-Derived Microparticles, Cluster Analysis, Exosomes, Female, Flow Cytometry, Glycosylation, Humans, Lectins, Male, Polycystic Kidney, Autosomal Dominant, Protein Array Analysis, Protein Binding, Renal Insufficiency, Chronic, Urine, Uromodulin, Young Adult
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.

DOI10.1371/journal.pone.0074801
Alternate JournalPLoS ONE
PubMed ID24069349
PubMed Central IDPMC3777961
Grant List5RC1DK086161-02 / DK / NIDDK NIH HHS / United States
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