RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues

Abstract

Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step.

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

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Selective Binding of Heparin/Heparan Sulfate Oligosaccharides to Factor H and Factor H-Related Proteins: Therapeutic Potential for C3 Glomerulopathies

“…Glycosaminoglycan extractions were performed as described previously ( 30 ). GAGs were loaded on 1% (w/v) low-melting agarose gel (Boehringer Mannheim, Mannheim, Germany), and resolved by gel electrophoresis in 50 mM barium acetate buffer, pH 5.0 (Sigma-Aldrich).…”

Glomerular endothelial glycocalyx-derived heparan sulfate inhibits glomerular leukocyte influx and attenuates experimental glomerulonephritis

“…The upper layer (aqueous phase) was dialyzed against 5 × 5 L baths of Milli-Q H 2 O using SnakeSkin dialysis membranes (MWCO 3500 Da, Thermo Scientific) and dried using a Savant SC210A SpeedVac concentrator (Thermo Scientific). To isolate the individual GAG constituents, mGEnC glycocalyx was separated on 1% agarose gel in barium acetate ( van de Lest et al, 1994 ; van Gemst et al, 2016 ), followed by excision and phenol extraction of the separated HS and CS. Individual fractions were ethanol-extracted several times to remove phenolic contamination.…”