Heparin's roles in stabilizing, potentiating, and transporting LEDGF into the nucleus

PURPOSE. Lens epithelium-derived growth factor (LEDGF) is a 60-kDa protein that dramatically enhances cellular survival, growth, adhesiveness, and res istance to heat and oxidative stress. Full-size recombinant LEDGF is degrad ed during prokaryotic preparation. Heparin's capacity to stabilize recombin ant LEDGF in the face of various stresses (heat, pH, proteolysis), to poten tiate its growth-enhancing properties, and to enable transport of LEDGF int o the nucleus of mouse lens epithelial cells has been characterized. METHODS. LEDGF-cDNA was cloned in a pGEX-2T expression vector to produce a fusion protein, GST-LEDGF. Porcine heparin was used to stabilize GST-LEDGF. Heparin-Sepharose was used to characterize heparin-GST-LEDGF binding, and GST-LEDGF or heparin-GST-LEDGF was used to quantitate heparin's stabilizati on of LEDGF in the face of heat, pH, and proteolytic stresses. Fluorescein isothiocyanate-labeled GST-LEDGF and heparin-GST-LEDGF were incubated With cultured mouse lens epithelial cells (LECs). Fluorescence microscopy and im munostaining techniques were used to monitor heparin's potentiation of LEDG F's growth stimulation and heparin's role in. the translocation of GST-LEDG F from the extracellular space into the cytoplasm and nucleus. RESULTS. Heparin, at concentrations as low as 7.1 mg/ml, protected GST-LEDG F from degradation and increased the yield of the full-size fusion protein in a prokaryotic system. It also protected GST-LEDGF from heat, acid-base d eactivation, and proteolytic degradation with trypsin and chymotrypsin and greatly potentiated LEDGF's enhancement of mouse LEC growth in culture. It also increased nuclear uptake of exogenous GST-LEDGF and endogenous LEDGF. CONCLUSIONS. Heparin protected GST-LEDGF from degradation under various str ess conditions and facilitated transport of GST-LEDGF into the nucleus.