Mutational Analysis of the Lens Intrinsic Membrane Protein MP19/To3 Cataract Locus

Abstract

Abstract: : Purpose: MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific mutation of MP19 at amino acid 15 (G15V), termed MP19^To3 leads to total heritable cataract and microphthalmia. Further studies have shown that normal MP19 transported to the cell membrane while MP19^To3 did not. It appeared that MP19^To3 was trapped in the pre-Golgi (ERGIC). The goal of this study was to determine whether other mutations at amino acid 15 results in the MP19^To3 phenotype or the normal MP19 phenotype. Methods: MP19 cDNA point mutations were generated using the QuickChange site-directed mutagenesis kit sold by Stratagene. The mutations were inserted into a tetracycline-inducible mammalian expression vector containing MP19 cDNA and EGFP coding cDNA (MP19 cDNA-EGFP-pcDNA4.0/TO vector). Upon induction, the vector directs the synthesis of MP19/EGFP fusion protein. DNA sequencing was performed on each of the constructs to confirm the point mutations. Several different amino acid substitutions were made at amino acid 15, including proline, alanine, asparagine, glutamine, phenylalanine, and aspartic acid. Each of the mutation plasmids was transfected into human TRx-293 cells using FuGene 6. Fluorescent expression of the MP19 fusion protein was viewed using confocal microscopy. Proteins from the different mutants, including normal MP19 and MP19^To3 were isolated using different extraction methods, separated using SDS-PAGE, and then western blots were analyzed to determine the localization of the proteins in the cells. Results: Each of the mutant plasmids expressed fluorescent protein differently in cultured cells. Alanine-EGFP-pcDNA4.0/TO was observed to transport to the cell membrane very similar to normal MP19-cDNA-pcDNA4.0/TO. Phenylalanine-EGFP-pcDNA4.0/TO appeared to sequester in the ERGIC, very similar to MP19^TO3. The proline substitution appeared to traffic the MP19-Proline-EGFP molecule to an area just under the cell membrane, while the asparagine and glutamine mutations appeared to sequester in very small "pools" either in the cell membrane or just under the membrane. Conclusions: Each of the different amino acid mutations at amino acid 15 of the MP19 molecule appeared to have a specific and different influence on trafficing of MP19 to the cell membrane.

Keywords: protein structure/function • molecular biology • proteins encoded by disease genes