Identification of a lens-specific cis-acting element within the basal promoter of the human lens intrinsic membrane protein MP19 gene (LIM2).

Using overlapping and mutant oligonucleotides as probes, gel mobility assays and competition experiments identified a sequence from -47 to -32 bp upstream of the LIM2 CAP site, which a lens protein complex bound with high affinity which appeared to bind only to the "sense" strand of the double-stranded DNA molecule. This sequence consisted of a string of four guanine residues followed by seven other nucleotides (AACCTAA) and followed by another four guanines, i.e. GGGGAACCTAAGGGG, called the Hsu element. Promoter-CAT constructs containing this sequence or mutations of the sequence indicated that the Hsu element is located within the basal promoter, and is essential for expression of the LIM2 gene. The trans factors binding to the Hsu element are present throughout development, and appear to be lens-specific. Since the LIM2 gene promoter does not contain a classic TATA box, the Hsu element may serve as the site for binding the RNA polymerase complex.

Does lens intrinsic membrane protein MP19 contain a membrane-targeting signal?

PURPOSE: Lens intrinsic membrane protein MP19 is the second most abundant major protein of the lens fiber cell membrane and appears to be specific to the lens. Different mutations of this protein are known to cause cataract in both humans and mice. To date, the function of MP19 in the lens is not known, nor is the mechanism by which the protein migrates to the cell membrane. The goal of this study was to determine whether or not MP19 distributes to the cell membrane directed by a peptide signal within the sequence of the molecule. METHODS: Using PCR, MP19 cDNA was truncated to yield separate fragments coding for the first 25, 36, and 64 amino acids of the MP19 polypeptide chain. These PCR fragments were further cloned into mammalian expression vector pcDNA4/TO, a tetracycline-regulated vector that, upon induction with tetracycline, allows expression of cDNA inserts within the vector. These vectors expressed each of the MP19 truncated fragments fused to EGFP. Each of the prepared plasmids was transfected into T-REx-293 cells using FuGene 6. Cloned cell lines from each of these transfections were obtained and used in the studies. The fluorescent expressed protein was viewed using confocal microscopy. Proteins from the different cell lines were isolated by different membrane extraction methods and western blot analysis was carried out to further determine the localization of expressed MP19 and MP19 truncated fragments. RESULTS: Cell lines expressing intact MP19/EGFP (with EGFP fused to the COOH-terminal end of MP19, MP19G) fusion protein were observed to traffic MP19 to the cell membrane, where it appeared to sequester in rather large pools. All of the MP19 truncations (with EGFP fused to the COOH-terminal end of each truncation; MP19-25G, MP19-36G, and MP19-64G) appeared to also traffic EGFP to the cell membrane. MP19-25G and MP19-36G did not distribute uniformly on the membrane, but appeared to localize into smaller, punctate "spots" of fluorescent material. MP19-64G distributed on the membrane similarly to MP19-25G and MP19-36G, however, the punctate areas of fluorescent material were considerably larger and similar to that demonstrated by intact MP19G. Western blot analysis of isolated total membranes, intrinsic membranes, and lipid rafts showed that MP19G and MP19-64G were associated with the intrinsic membrane fraction while MP19-25G and MP19-36G were at least 75% associated with the intrinsic membrane fraction. All of the preparations appeared to be at least 50% associated with membrane lipid rafts. However, when EGFP/MP19-25 and EGFP/MP19-36 (with EGFP fused to the NH2-terminal end of the truncated peptide, GMP19-25 or GMP19-36) were expressed, the fusion protein was observed to remain completely soluble in the cytoplasm, identical to expressed EGFP alone. Western blots of these two fusion proteins also indicated that the product did not associate with the cell membrane. In contrast, when EGFP/MP19 (with EGFP fused to the NH2-terminal end of intact MP19, GMP19) was expressed, the fusion protein did integrate into the cell membrane, identical to MP19G. Western blot analysis revealed that GMP19 also associated with lipid rafts, identical to intact MP19G. CONCLUSIONS: It appears that the first 25 amino acids of the MP19 molecule are sufficient to target the protein to the cell membrane, and apparently integrate into the membrane. With the addition of more amino acids, the polypeptide distributes in the membrane similarly to that of the intact MP19 molecule. It appears that the first 25 amino acids of the MP19 molecule is, indeed, a membrane signal and integration sequence. Also, at least part of these 25 amino acids must integrate into the cell membrane, but not extend through the cell membrane.

Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors.

PURPOSE: [corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19. METHODS: MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy. RESULTS: Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell. CONCLUSIONS: The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.

Temporal expression of three mouse lens fiber cell membrane protein genes during early development.

PURPOSE: Three different lens fiber cell intrinsic membrane proteins, MIP (Major Intrinsic Protein), MP19, and connexin50 (Cx50), have separately been implicated as causative candidates for congenital cataracts. The aim of this study was to determine gene transcript expression of these three proteins during successive stages of mouse embryonic development. METHODS: Total RNA was prepared from mouse embryos taken at days 9-10 (E9-E10) of gestation, heads of day 11-13 (E11-E13) embryos, and lenses of adult mice. Coupled reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene transcript expression of MIP, Cx50, and MP19 during embryonic development. The products of RT-PCR were further cloned into the TOPO(TM) TA vector, and further analyzed by double strand nucleotide sequencing. RESULTS: Cx50 gene expression was observed throughout the developmental period observed (E9-E13). MIP transcripts were first observed at mouse embryonic day 11.5 (E11.5) and synthesis continued throughout the developmental period observed. The gene for MP19 (Lim2) begins to express at mouse embryo day 12 (E12) and synthesis continued throughout the developmental period observed. mRNA levels for all three proteins appear to remain steady from these early embryonic stages through adulthood. CONCLUSIONS: The identified early expression of Cx50, MIP, and Lim2 transcripts in mouse embryonic stages suggests that all three proteins play very important, probably quite different, roles in lens fiber cell differentiation. Variation in the temporal expression of these three genes during the course of development suggests a critical gene-coordinated regulation throughout lens fiber cell development. These three genes clearly play important roles in early normal lens development since it is known that mutations in the sequence of each membrane protein results in cataractogenesis.