Heparin's roles in stabilizing, potentiating, and transporting LEDGF into the nucleus.
OBJECTIVE
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 nucleus.
The Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
N
N
Fatma
D P
DP
Singh
T
T
Shinohara
L T
LT
Chylack
D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C119380:lens epithelium-derived growth factor; D006493:Heparin; D005982:Glutathione Transferase