The project describes basic immunological and molecular biological studies of a lens-epithelium-derived growth factor (LEDGF), a protein possibly involved in the mechanism of age-related cataract (ARC), the leading cause of world blindness. In earlier publications, the PI showed that sera from patients with ARC contained antibodies (Abs) cytocidal for mouse and human lens epithelial cells (LECs) in culture. These cytocidal Abs were highly prevalent (96%) in patients with ARC. He began a search for the lens protein which could induce such a cytocidal Ab. From human LECs several clones were isolated using an auto-Ab probe from patients with ARC. One of these clones was isolated, purified, and its DNA sequence was determined. From this the amino acid sequence of LEDGF was deduced. LEDGF contains 530 amino acids and has a molecular weight was 61 kDa. A search of computerized data banks of primary protein structures revealed that LEDGF was highly homologous to hepatoma-derived growth factor. Its primary structure suggests that LEDGF may be a regulatory protein. LEDGF significantly stimulated the growth of LECs in culture. Anti-LEDGF Abs blocked this stimulatory effect, and cells incubated with anti-LEDGF Abs ultimately died. The seven specific objectives outline a plan to produce LEDGF, to define its secondary and tertiary structures, its tissue expression and its function in cell growth and differentiation. The PI will determine the amino acid sequence of its epitopic region and its antigenic potency to induce cytocidal anti-LEC Abs. He will isolate the LEDGF gene and produce a knockout mouse to further our assessment of the functions of LEDGF. In a mouse model he will define the age-dependency of LEDGF's effect on LECs. Also in a pilot clinical study the PI will recruit age- and sex-matched patients with clear lenses and ARC, do LOCs III-cataract classification, and anti-LEDGF Ab titers to determine the significance of any association with a particular class of ARC. The discovery of a lens-derived growth factor, an Ab which blocks its stimulatory effect, and the high prevalence of this Ab in patients with ARC comprise and immunological system which may modify the integrity of the lens epithelium, the metabolic """"""""engine"""""""" of the lens, and cause age-related cataract.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences A Study Section (VISA)
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Brigham and Women's Hospital
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Kubo, Eri; Singh, Dhirendra P; Fatma, Nigar et al. (2003) Cellular distribution of lens epithelium-derived growth factor (LEDGF) in the rat eye: loss of LEDGF from nuclei of differentiating cells. Histochem Cell Biol 119:289-99
Nishizawa, Y; Usukura, J; Singh, D P et al. (2001) Spatial and temporal dynamics of two alternatively spliced regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and p52, in the nucleus. Cell Tissue Res 305:107-14
Nakamura, M; Singh, D P; Kubo, E et al. (2000) LEDGF: survival of embryonic chick retinal photoreceptor cells. Invest Ophthalmol Vis Sci 41:1168-75
Singh, D P; Ohguro, N; Kikuchi, T et al. (2000) Lens epithelium-derived growth factor: effects on growth and survival of lens epithelial cells, keratinocytes, and fibroblasts. Biochem Biophys Res Commun 267:373-81
Sharma, P; Singh, D P; Fatma, N et al. (2000) Activation of LEDGF gene by thermal-and oxidative-stresses. Biochem Biophys Res Commun 276:1320-4
Fatma, N; Singh, D P; Shinohara, T et al. (2000) Heparin's roles in stabilizing, potentiating, and transporting LEDGF into the nucleus. Invest Ophthalmol Vis Sci 41:2648-57
Singh, D P; Kimura, A; Chylack Jr, L T et al. (2000) Lens epithelium-derived growth factor (LEDGF/p75) and p52 are derived from a single gene by alternative splicing. Gene 242:265-73
Balaram, M; Tung, W H; Kuszak, J R et al. (2000) Noncontact specular microscopy of human lens epithelium. Invest Ophthalmol Vis Sci 41:474-81
Ayaki, M; Sueno, T; Singh, D P et al. (1999) Antibodies to lens epithelium-derived growth factor (LEDGF) kill epithelial cells of whole lenses in organ culture. Exp Eye Res 69:139-42
Singh, D P; Ohguro, N; Chylack Jr, L T et al. (1999) Lens epithelium-derived growth factor: increased resistance to thermal and oxidative stresses. Invest Ophthalmol Vis Sci 40:1444-51