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Nigar Fatma, Dhirendra P. Singh, Toshimichi Shinohara, Leo T. Chylack; Heparin’s Roles in Stabilizing, Potentiating, and Transporting LEDGF into the Nucleus. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2648-2657.
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purpose. Lens epithelium-derived growth factor (LEDGF) is a 60-kDa protein that
dramatically enhances cellular survival, growth, adhesiveness, and
resistance to heat and oxidative stress. Full-size recombinant LEDGF is
degraded during prokaryotic preparation. Heparin’s capacity to
stabilize recombinant LEDGF in the face of various stresses (heat, pH,
proteolysis), to potentiate its growth-enhancing properties, and to
enable transport of LEDGF into 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 stabilization 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 immunostaining techniques
were used to monitor heparin’s potentiation of LEDGF’s growth
stimulation and heparin’s role in the translocation of GST-LEDGF from
the extracellular space into the cytoplasm and nucleus.
results. Heparin, at concentrations as low as 7.1 mg/ml, protected GST-LEDGF
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 deactivation, 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 stress
conditions and facilitated transport of GST-LEDGF into the
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