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.

N-linked (N-) Glycoproteomics of Urimary Exosomes*

“…However, glycosylation of exosomal proteins is widely unknown with the exception of classes of glycans on the surface as evidenced by lectin binding assays. This surface glycome is known to change in autosomal dominant polycystic kidney disease compared with healthy volunteers' urinary exosomes (30). In this study, we have characterized multiple glycoproteins, their N-glycosylation site, glycan compositions, and structures.…”

Extracellular Vesicles in Renal Diseases

“…90 These proteins and others, such as Cystin and ADP ribosylation factor-like 6, have been found in uEX of patients with PKD. 13,27 Another approach to find a novel diagnostic/monitoring tool for PKD was the study of lectin profiles of uEVs, 91 showing that lectin microarray profiles of uEVs of patients with PKD and healthy controls have different patterns. This suggests possible diseasespecific modifications.…”

Isolation of Urinary Extracellular Vesicles from Tamm- Horsfall Protein–depleted Urine and their Application in the Development of a Lectin-Exosome-Binding Assay

“…To overcome this problem, THP depolymerization by DTT (dithiothreitol) treatment (18) or uEV isolation by sucrose gradient centrifugation (15,16,19,20) have been routinely used, but both approaches have disadvantages (12,16,21,22). Even when DTT is used, THP can still be present in the isolated uEVs (15,21,23,24), whereas separation on sucrose gradients or cushions (15,19,20) is time-consuming, taking up to 24 hours (15,16,19,20).…”