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.
Highlights
Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides and prominent components of extracellular matrices and cell surface glycan layers
To isolate and characterize GAGs expressed by mGEnC-1, a published protocol for the isolation of GAGs from tissues [23, 24] was followed
Isolated GAGs were quantified by an adapted 1,9-dimethylmethylene blue (DMMB) GAG quantification assay based on the Farndale method [23,24,25], which relies on the formation of a GAG-cationic dye complex
Summary
Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides and prominent components of extracellular matrices and cell surface glycan layers. The sequence of modifications along the carbohydrate backbone allows sulfated GAGs, HS, to bind growth factors, chemokines and cellular adhesion molecules, such as fibroblast growth factors, interleukin-8, selectins and the macrophage-1 antigen (Mac-1), thereby regulating various physiological processes including cell growth, morphogenesis, coagulation and inflammation [1,2,3,4,5,6,7,8,9]. We have isolated and characterized a unique mouse glomerular endothelial cell line (mGEnC-1) [13] and identified sulfated HS domains in the glomerular endothelial glycocalyx that mediate chemokine binding and leukocyte trafficking during inflammation in vitro and in vivo [13,14,15,16,17,18]. Since the presence of many additional functional GAG domains in the glomerular endothelial glycocalyx is presumed, isolation and analysis of intact GAGs, e.g. using mass spectrometry, may yield novel structural information about functional GAG domains
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
