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Profiling proteoglycans: RIP them out

Functional Glycomics (13 August 2009) | doi:10.1038/fg.2009.27

A new approach for the quantitative isolation of proteoglycans facilitates glycomics profiling and functional studies.

A simplified scheme of the assembly and extension of HS-GAG chains on a PG core protein. Kidney International (2008) 74, 289–299; doi:10.1038/ki.2008.159

The fine structure of heparan sulfate (HS) influences protein binding and regulation by heparan sulfate proteoglycans (HSPGs). Cell-surface or extracellular HSPGs are present in all animals and have complex roles, thus there is a need to define how variations in HS structure contribute to the specificity of HSPG interactions with other proteins. Existing methods for purifying HS are long and involved, requiring relatively large amounts of starting material and often resulting in non-native extracts that are unsuitable for use in bioassays. Reporting in the Journal of Biological Chemistry, Turnbull and colleagues describe a new method for the rapid isolation of proteoglycans (RIP) that addresses these limitations when combined with sensitive compositional analysis. This new technique aids the investigation of HS function in vivo and improves understanding of proteoglycan biology.

Similar to other glycosaminoglycans (GAGs), HS is a linear molecule, formed from a repeating disaccharide unit, which is assembled on a core protein during proteoglycan synthesis. The basic HS unit consists of glucuronic acid and N-acetylglucosamine (UA-GlcNAc). However, numerous Golgi enzymes work to sulfate and epimerize regions of the HS chain, resulting in varied structures that are further modified by cell-surface sulfatases. Improving the purification of HSPGs, and the sensitivity of their structural analyses, allows researchers to define which HS structures are responsible for specific protein binding and which are expressed in different tissues, giving an insight into their function.

The RIP method is based on a common RNA extraction procedure where HSPGs partition to the aqueous phase during TRIzol (Invitrogen) extraction of cells and tissues. The initial isolate was further purified by anion exchange chromatography and enzymatic digestion, resulting in a highly purified HS preparation after approximately 24 hours. High yields of spiked standards were obtained, and NMR analysis confirmed that the RIP procedure does not alter the structure of the polysaccharides.

After extraction, the HS composition was analyzed using a highly sensitive labeling and detection method recently developed by the authors. HS disaccharides resulting from complete heparinase digestion were labeled with the fluorophore BODIPY hydrazide, separated using high-performance anion exchange chromatography and detected with laser-induced fluorescence. The typical HS profile had a high proportion of the unsulfated UA-GlcNAc with smaller peaks for the variant disaccharides; the proportions differed for specific tissues.

The RIP-BODIPY method is sensitive enough to detect low-abundance HS disaccharides from only a few thousand cultured cells, or a several hundred micrograms of wet tissue, facilitating the study of knockout mouse models. The authors separately investigated mice deficient in sulfatase enzymes 1 and 2 (Sulf-1 and Sulf-2). The HS profiles of the mice differed, indicating the divergent action of these enzymes in vivo. The HS profile of Sulf-1 knockout tissue also revealed a preference for Sulf-1 acting on trisulfated disaccharides, which differed from the profile observed with cultured Sulf-1 knockout cells, therefore emphasizing the importance of in vivo studies.

Functional studies are made possible because RIP-purified HS retains its structural integrity. HS binding to fibroblast growth factor 2 (FGF2) requires 2-O-sulfate groups, which are added by 2-O-sulfotransferase (2OST). In contrast to HS from wild-type mice, HS from 2OST-/- mice failed to activate FGF2 signaling by FGFR1. Surprisingly however, HS from 2OST+/- mice was also inactive in the FGF signaling bioassay, indicating that 2OST gene dosage is critical for HS-dependent FGF signaling.

The HS profiles revealed in this study, and the divergent signaling capacities of different HS structures, indicate how important it is to profile proteoglycans. The RIP–BODIPY method will complement other advances, including improved mass-spectrometry-based methods, towards a complete understanding of proteoglycan function.

Emma Leah

Original research paper:

  1. Guimond, S. E. et al. Rapid purification and high sensitivity analysis of heparan sulfate from cells and tissues: towards glycomics profiling. J. Biol. Chem. (published online 13 July 2009) doi:10.1074/jbc.M109.032755. | Article |