Heparan Sulfate Proteoglycan-Mediated Polyamine Uptake
The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display–derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.
Key wordsHSPG Polyamine uptake Flow cytometry analysis CHO-cells Anti-HS antibodies Heparinase III lyase treatment EXT-1 knock-out cells
This work was supported by grants from The Swedish Cancer Fund; The Swedish Research Council; The Swedish Society of Medicine; The Physiographic Society, Lund; The Crafoordska, Gunnar Nilsson, and Kamprad Foundations; and the Lund University Hospital (ALF).
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