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.

Fig 1. Characterization of mGEnC-1 GAGs by barium acetate gel electrophoresis and DMMB analysis.

The GAG content in extracts from conditionally immortalized mouse glomerular endothelial cells (mGEnC-1) was visualized by barium acetate gel electrophoresis (A), and quantified relative to heparan sulfate from bovine kidney using 1,9-dimethylmethylene blue (B). Analysis of heparinase I, II and III-treated mGEnC-1 GAGs on gel indicated no degradation of the spot that co-migrates with the HS standard.

Fig 2. Characterization of mGEnC-1-derived GAGs reveals RNA as a major contaminant.

The RNA content in extracts from conditionally immortalized mouse glomerular endothelial cells (mGEnC-1) was visualized by ethidium bromide agarose gel electrophoresis (A), and barium acetate gel electrophoresis (B) before and after RNase treatment. Enzymatic degradation of RNA in mGEnC-1 GAG extracts removes the RNA band observed on ethidium bromide gel, and a large spot that appears to co-migrate with HS on the barium acetate gel.

Fig 3. Glycosaminoglycans (GAG) extracts contain significant RNA impurities, which can be digested by RNase-I treatment.

Visualizing nucleotide impurities in GAG preparations from conditionally immortalized mouse glomerular endothelial cells (mGEnC-1), human umbilical vein endothelial cells (HUVEC), immortalized retinal pigmental epithelial cells (ARPE-19) (A), and mouse kidney, heart, liver and spleen (B) on ethidium bromide agarose gels, revealed significant RNA contaminations. RNase-I treatment efficiently removed the contamination from mGEnC-1 and ARPE-19 GAGs, though minor impurities remained in HUVEC-, kidney- and spleen-derived extracts.

Fig 4. RNA impurities interfere with the analysis of glycosaminoglycans (GAGs) using barium acetate agarose gel electrophoresis.

Resolving untreated GAG extracts by barium acetate gel electrophoresis suggested relatively large amounts of heparan sulfate (HS) and dermatan sulfate (DS) and relatively little chondroitin sulfate (CS) in GAGs obtained from mouse glomerular endothelial cells (mGEnC-1), human umbilical vein endothelial cells (HUVEC) and immortalized retinal pigmental epithelial cells (ARPE-19) (A). GAG extracts from mouse tissues appeared to contain large amounts of DS (heart and liver) and CS (spleen), whereas kidney-derived GAGs were enriched in HS, but also contained DS and CS (B). However, the observed staining patterns seemed to result from contaminating RNA co-migrating between HS and DS, as RNase-I treatment revealed the actual GAG spots corresponding to primarily HS and CS.

Fig 5. RNA contamination of glycosaminoglycan (GAG) extracts leads to a significant overestimation of GAG yields.

The GAG content in extracts from conditionally immortalized mouse glomerular endothelial cells (mGEnC-1), human umbilical vein endothelial cells (HUVEC), immortalized retinal pigmental epithelial cells (ARPE-19) (A) and C57BL/6J mouse kidney, heart, liver and spleen (B) was quantified relative to heparan sulfate from bovine kidney using 1,9-dimethylmethylene blue. The apparent yield in untreated GAG samples from cell cultures was significantly overestimated 2- to 6-fold compared to RNase-treated samples, indicating that RNA contamination interferes with the charge-based DMMB quantification method. RNA also interfered with the quantification of GAGs obtained from heart, liver and spleen, but not in kidney cortex extracts. GAG concentrations are presented as μg/cm² confluent cell monolayer or μg/mg wet tissue. Results are given in means ± s.e.m. *P<0.05 by Anova.

Fig 6. Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment

Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).