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A.K. Kundzewicz, F.L. Munier, J.–M.M. Matter; Expression and Cell Compartmentalization of EFEMP1, a Protein Associated With Malattia Leventinese . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3113.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A single missense mutation in EGF–containing fibrillin–like extracellular matrix protein 1 ( EFEMP1 ) is associated with Mallatia Leventinese, an inherited macular degenerative disease. Wild type protein is known to be secreted whereas mutant is misfolded and retained within cells. Cells expressing EFEMP1 gene have not been identified so far, which promped us to study the expression pattern and the cell compartmentalization of EFEMP1 proteins. As mutation may alter secretion of this protein we compared cell compartmentalization of normal and mutated proteins. Methods: To determine expression patterns of the EFEMP1 gene, in situ hybridization studies were carried out on paraffine sections of normal and disease ( ML and AMD ) human retinas. Fusion proteins containing wild type and mutated ( R345W ) EFEMP1 ( short and long splice variants ) with C–terminal GFP or flag–peptide under the control of the CMV promoter were constructed in order to determine cellular localization of the protein. Plasmids were then transfected into different cell lines ( VERO – African green monkey kidney, COS7, CHO ) by using Lipofectin (GIBCO). In all transfection experiments 0,8 µg of plasmid DNA was used per 40’000 cells. After 12–48 hours cells were analyzed in situ or collected for Western–blot analysis. Results: In normal eye EFEMP1 transcripts are localized mainly in a subclass of retinal ganglion cells and in the majority of photoreceptors. In the diseased human eye, robust accumulation of transcripts is principally found in the RPE accompanied with drastic morphological changes of this tissue. This upregulation is not specific for the ML eye except in the ciliary body. Fusion protein studies performed on VERO cells revealed the localization of EFEMP1–GFP and ML–GFP proteins. While long variants of the protein showed a uniform, cytoplasmic localization, short variants were localized within vacuoles and the cells expressing the transgenes died few hours after transfection. Western–blot analysis showed that the long variant of the protein is stable and gives a strong signal whereas the short variant is degraded. Conclusions: Morphological alterations of RPE cells in ML eye coincide with significant changes in the expression pattern of the EFEMP1 gene. The specificity of EFEMP1 expression in the ML ciliary body is striking and sets the difference between ML and both normal and AMD eyes. Compartmentalization of short versus long variants differs. Secretion of both splice variants in different cell lines as well as the comparison of their gene expression patterns are currently under investigation.
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