|Molecular Vision 2003;
Received 18 November 2003 | Accepted 21 December 2003 | Published 22 December 2003
Does lens intrinsic membrane protein MP19 contain a membrane-targeting signal?
XiaLian Li, Yu
Yang, Agiimaa Gan Erdene,
Robert L. Church
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA
Correspondence to: Robert L. Church, Emory Eye Center, Room B5601, 1365B
Clifton Road, NE, Atlanta, GA, 30322; Phone: (404) 778-4101; FAX: (404)
778-2232; email: email@example.com
Dr. Li is now at the Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, Peoples Republic of China
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.
MP19 (referred to elsewhere as MP17, MP18, and MP20 [1-3]) is the second most abundant integral membrane protein in lens fibers. It was first described as a fiber-specific component of bovine lens membranes  and a cDNA encoding this protein was subsequently cloned from a bovine lens cDNA library . A rat cDNA  and the entire human and mouse genes [5,6] have since been cloned and reported.
As the second most abundant lens membrane protein, MP19, like MIP, has been proposed to be a lens junction protein . The N-terminal amino acid sequence of MP19 is highly conserved among a number of mammals ( and unpublished) and appears to have lens-specific expression [1,2]. MP19 is phosphorylated by cAMP-dependent protein kinase and appears to bind calmodulin [1,7,8] and galectin-3 [9,10]. These features are indicative of a protein with a function that can be modulated. However, MP19 bears no striking resemblance to any other reported protein and has no clearly defined structural or functional role. However, like MIP, immunolocalization studies have identified MP19 in both junctional and non-junctional lens regions of fiber cell plasma membranes [7,11]. It is therefore possible that MP19 may play some role in gap junction formation, maintenance, or organization. MP19 has a very limited amino acid sequence homology with members of the EMP (epithelial membrane protein)/PMP22 (peripheral myelin protein 22) family (not more than 29% identity over select sequences, ). However, the significance of this very limited homology is questionable. Recently, Grey et al.  determined that MP19 resides in the cytoplasm of nucleated peripheral fiber cells, only becoming inserted into the cell membrane following degradation of fiber cell nuclei. They also determined that this protein migration event coincided with the creation of a barrier between fiber cells, restricting the movement of molecules through the lens by way of the extracellular space. This would indicate that MP19 might function as a cell adhesion molecule.
The updated topological model for MP19 in the lens fiber cell plasma membrane has been proposed to include two α-helical and two β-strand transmembrane segments  based upon the previously described cDNA-derived amino acid sequence [2,4], with the first 25, highly hydrophobic, amino acids being located on the cytoplasmic side of the membrane (Figure 1). However, the precise role that MP19 plays in the lens remains to be determined.
The mouse Lim2 gene, encoding MP19, has been recently isolated and characterized . In the mouse, Lim2 maps to chromosome 7 , and in the human, LIM2 maps to chromosome 19 (19q13.4) [5,16]. Shortly after the murine Lim2 gene was assigned, the heritable To3 cataractous mouse mutant was linked to the same chromosomal region as Lim2 , making the Lim2 gene (encoding the MP19 protein) an ideal candidate gene for the cataract . To3 heterozygous mutant mice display congenital, hereditary, bilateral total opacities of the lens, and homozygous animals display a total opacity of the lens, compounded by microphthalmia. This mutation is characterized by a single G->T transition mutation within the first coding exon (exon 2) of the Lim2 gene, resulting in the substitution of the normally encoded glycine by valine at amino acid 15 within the polypeptide sequence. This cataract was subsequently reproduced in transgenic mice by mutating the normal Lim2 gene with the single-base transversion, as was found in the To3 mice. Again, the identical total cataract was observed, compounded by microphthalmia . Thus these transgenic mice have the same dominant gain of function in their phenotype. The G->T mutation at amino acid 15 in the To3 cataractous mouse indicated that this mutation may result in an altered interaction of this region of MP19 with some cellular component rather than an altered insertion of this protein into the fiber cell membrane as was suggested by Steele, et al. . However, more recent evidence utilizing green fluorescent protein (GFP) expression vectors  demonstrates that MP19To3 does not traffic to the cell membrane, rather it appears to become trapped in the cell's endoplasmic reticulum Golgi intermediate complex (ERGIC). These data indicated that the first several amino acids in MP19 might play an important role in directing the MP19 protein to the cell membrane. A possible mechanism for the failure of MP19To3 protein to migrate to the cell membrane would involve an alteration of some normal signal that directs MP19 to the membrane; instead the mutation alters the normal signal, resulting in localization of MP19To3 to a subcellular compartment. Another possible mechanism that might cause MP19 to collect in the ERGIC would be that the point mutation might cause protein misfolding or aggregation within the cell, thus leading to cataract.
The goals of this study were to further determine the pathway of MP19 transport to the cell membrane and determine whether MP19 migrates to the cell membrane and integrates into the membrane by use of a signal.
Production of MP19 truncated gene fragment mammalian expression vectors
We designed three different sets of oligonucleotide primers, all of which had the same forward primer and each of the reverse primers were at different locations in the MP19 cDNA. As a template, we used mouse MP19 cDNA (AF320075). All of the polymerase chain reaction (PCR) truncations shared the same forward primer that contained a Hind III site just upstream of the MP19 ATG start codon. All of the reverse primers contained a BamH I site at the 3' end and were located at the 25th amino acid, the 36th amino acid, and the 64th amino acid in the MP19 protein. Each of the different sized truncation fragments were amplified by PCR. Temperature cycling was 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 2 min, over a total of 36 cycles. The PCR products were run in 1% agarose and fragments were cut out from the gel and purified using a Gel Extraction Kit (Qiagen, Valencia, CA).
Each of these truncation products was initially inserted into the TOPO TA Cloning Kit (pCR®4-TOPO vector, Invitrogen, Carsbad, CA) in order to obtain complete sequence analysis of the MP19 products and to facilitate further molecular manipulations. Then the MP19 products were removed from pCR®4-TOPO by restriction digestion with Hind III and BamH I and cloned into the multiple cloning site (MCS, at the Hind III and BamH I sites) of vectors pEGFP-N1 and pEGFP-C2, from the Living Colors Fluorescent Protein Reporter System (CLONTECH, Palo Alto, CA). The pEGFP-N1 and pEGFP-C2 vectors expressed green fluorescent protein upon transfection into mammalian cells. The MCS of pEGFP-N1 is located between the immediate early promoter of CMV and the EGFP coding sequence, and the MCS of pEGFP-C2 is located immediately after the EGFP coding sequence (Figure 2). MP19 and each of the separate truncation fragments thus were expected to be expressed as fusions to the NH2-terminus (MP19G) or COOH-terminus (GMP19) of EGFP if they are in the correct reading frame and there are no intervening stop codons.
Subsequently, each of the MP19 truncation fragments or intact MP19, previously cloned into the pEGFP-N1 and pEGFP-C2 vectors (above) was cut out of these vectors, with the fluorescent (green, EGFP) protein coding region included. Hind III and Not I were used to cut out MP19 and the different truncation fragments with EGFP fused to the COOH-terminus from pEGFP-N1 (MP19G, MP19-25G, MP19-36G, and MP19-64G) and then were cloned into another vector, pcDNA4/TO, as outlined in . Nhe I and Xba I were used to cut MP19 and the different truncation fragments with EGFP fused to the NH2-terminus from pEGFP-C2 (GMP19, GMP19-25, and GMP19-36). Before using Nhe I and Xba I to cut the pEGFP-C2 constructs, these plasmids were transformed into dam- competent cells (One Shot INV 110; Invitrogen) to unmethylate the Xba I site. Nhe I and Xba I have compatible sticky ends (3'-GATC-5'). The pcDNA4/TO vector was cut by Xba I and ligated with the Nhe I/Xba I fragments that were cut from the constructs of pEGFP-C2. Figure 2 shows a cartoon of both of the expression vectors and their product designations. BamH I was used to confirm the correct orientation of the ligation products. The pcDNA4/TO vector (Invitrogen) contains two tetracycline operator 2 (TetO2) sites within the human cytomegalovirus immediate-early (CMV) promoter for tetracycline-regulated expression of the gene of interest. The Tet repressor is expressed from the pcDNA6/TR plasmid. In the absence of tetracycline, expression of MP19 or MP19 truncations is repressed by the binding of Tet repressor homodimers to the TetO2 sequences. Addition of tetracycline to the cells derepresses the hybrid CMV/TetO2 promoter in pcDNA4/TO and allows expression of the various fusion proteins.
Cell culture, plasmid transfection, and generation of stable cell lines
Cell line T-REx-293, a human transformed primary embryonal kidney cell line (CLONTECH, Palo Alto, CA) was used for transfections of MP19 and MP19 truncation vectors. This cell line stably expresses the tetracycline (Tet) repressor. The newly generated MP19 truncated gene fragment expression plasmids were transfected into T-REx-293 cells using FuGene 6 (Roche Diagnostics Corporation, Indianapolis, IN). Stable clones of MP19-25-green-pcDNA4/TO (MP19-25G), MP19-36-green-pcDNA4/TO (MP19-36G), MP19-64-green-pcDNA4/TO (MP19-64G), MP19-green-pcDNA4/TO (MP19G), green-MP19-25-pcDNA4/TO (GMP19-25), green-MP19-36-pcDNA4/TO (GMP19-36), and green-MP19-pcDNA4/TO (GMP19) were picked and isolated by addition of 200 mg/ml zeocin to DMEM media. Each of the cloned expression cell lines was seeded onto glass coverslips or into tissue culture dishes. The expression of recombinant protein was induced by adding 5 μl of a 1 mg/ml aqueous solution of tetracycline to the culture medium. Cells expressing fluorescent EGFP fusion protein were viewed using confocal and fluorescent microscopy exactly as outlined in  or by western blot analysis.
DNA sequence analysis of fusion vectors
Primers were designed based upon the sequence of the expression plasmid pcDNA4/TO and were used to sequence the complete inserts from both directions in order to determine with complete assurance that the amino acid sequence of the expressed proteins was correct and not altered during the PCR and cloning process.
Vector DNA containing the fusion construct was used as a template in cycle sequencing reactions using Cy5-labeled oligonucleotide primers and the Cy5 AutoCycle Sequencing Kit (Pharmacia Biotech, Piscataway, NJ). These reactions were then electrophoresed and analyzed using an ALFexpress automated DNA sequencer (Pharmacia Biotech). DNA sequences were analyzed for identity with authentic mouse MP19 cDNA (AF320075) sequences using BLAST2 at NCBI (allowing comparison of our generated sequence to that of the MP19 cDNA GenBank sequence), and DNASIS sequence analysis software for Windows (Hitachi Software, San Bruno, CA).
Biochemical analysis of expressed MP19-EGFP and truncation fragments
Hypotonic lysis: Stable T-REx-293 cell lines transfected separately with various MP19 and truncation constructs were grown to confluence in 60 mm dishes and the synthesis of fusion protein induced with tetracycline. After three days of induction, the cells were scraped into cold PBS and pelleted by centrifugation at 1,000x g for 5 min. The pellets were then resuspended in 750 μl of hypotonic buffer (5 mM Tris, pH 7.5, 1 mM MgCl2, 1 mM EGTA, and 0.1 mM EDTA) containing protease inhibitors (leupeptin and aprotinin, 10 μg/ml each). After 30 min incubation on ice, the lysate was passed though a 26-gauge needle (10 times) and centrifuged at 1,000x g for 10 min to remove nuclear debris. The postnuclear lysate was transferred to an 11x35 mm (1.5 ml) polyallomer tube and centrifuged at 57,000 rpm for 30 min in a SW60 Ti rotor (Beckman). The supernatant was collected, and the pellet was resuspended in 300 μl of 1% SDS. Protein concentration was determined in both supernatant and pellet and equal quantities of protein (about 10 μg) from the samples were subjected to SDS-PAGE and western immunoblot analysis. This preparation separates proteins that are associated with cell membranes from those that are found soluble in the cytoplasm.
Alkaline carbonate extraction: A more stringent method for examining the high-affinity association of proteins with membranes is to expose cell membranes to high pH. Those proteins resistant to extraction are considered "integral membrane proteins" . Cell lines were grown to confluence in 60 mm diameter dishes and induced with tetracycline. Three days later, the cells were washed twice in ice-cold PBS and once in 150 mM NaCl. After aspiration of the NaCl solution, 1 ml of 100 mM NaCO3 (pH 11.3) containing protease inhibitors was added and the cells were scraped off the dish. The sample was transferred to a tightly fitting 1 ml Dounce homogenizer and homogenized (5 strokes). Following a 30 min incubation on ice, the sample was transferred to an 11x35 mm (1.5 ml) polyallomer tube and centrifuged at 57,000 rpm in a SW60 Ti rotor for 50 min. The pellet was resuspended in 300 μl of 1% SDS. Both the supernatant and pellet fractions were sonicated on ice, and equal quantities of protein (about 10 μg) from each sample were subjected to SDS-PAGE and western immunoblot analysis. This preparation typically separates proteins that are integrated into the membrane with transmembrane loops versus soluble cytoplasmic and membrane associated proteins. Methods very similar to the above have been used for many years to isolate lens intrinsic membrane proteins from intact lenses [22,23].
Triton solubility: Lipid rich microdomains of the cell membrane are often found in many cell types. These "lipid rafts" usually sequester membrane proteins that function in endocytosis and cellular signaling. The protein composition of these lipid rafts can be characterized following treatment of the cell membrane with a low percent Triton X-100 buffer at low temperatures. To investigate whether MP19 and the various truncations associate with lipid rafts, the different cell lines were grown to confluence in 60 mm dishes and induced with tetracycline for 3 days. The dishes were then washed twice with ice-cold PBS. Cold MBS (25 mM Mes [pH 6.5], 150 mM NaCl) containing 1% Triton X-100 plus protease inhibitors was added (300 μl). Following a 30 min incubation on ice, the cell material was pipetted up and down, and then centrifuged at 13,000x g for 15 min. The soluble fraction was collected from each of the cell lines and 300 μl of 1% SDS was added to the insoluble fraction. Then the insoluble fraction was passed through a 26-gauge needle (10 times) in order to lower its viscosity. Both fractions were subjected to SDS-PAGE and western immunoblot analysis. The triton-insoluble pellet contained membrane proteins that are associated with lipid rafts.
Samples were subjected to SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Membrane were then washed, blocked, incubated with primary antibody (anti-EGFP), washed again, and incubated with secondary antibody conjugated to alkaline phosphatase. The WesternBreeze chemiluminescent immunodetection system (Invitrogen) was used for immunodetection of EGFP fusion protein.
Results & Discussion
Intact MP19 protein with EGFP fused to the COOH-terminal end of MP19 (MP19G) was observed to transport to the cell membrane (Figure 3A,B), appearing to associate in "pools" or plaques on the surface of the cell membrane, reminiscent of proteins associated with lipid rafts. All of this class of MP19 truncations (MP19-25G, MP19-36G, and MP19-64G) also appeared to migrate to the cell membrane in a very similar manner as the intact MP19 molecule. MP19-25G (Figure 3C,D), MP19-36G (Figure 3E,F), and MP19-64G (Figure 3G,H) also appeared to associate with plaques on the cell surface.
Western blot analysis of each of the cell lines indicated that MP19G and all of the truncations (MP19-25G, MP19-36G, and MP19-64G) were membrane associated, as revealed by hypotonic lysis of the cells (Figure 4A-D, Hypotonic pellet). Upon alkaline extraction of the cells, which is an indicator of integral membrane proteins, MP19G and all of the MP19G truncations appeared to again associate completely with the membrane fraction (Figure 4A-D, Alkaline pellet), demonstrating that all of the peptide fragments were at least integrated into the cell membrane as integral membrane proteins. Further extraction of the cells with cold 1% triton-X100 revealed that At least 50% of the MP19G or MP19G truncation protein was associated with the insoluble fraction, that is, with lipid rafts (Figure 4A-D, Triton pellet), including the smallest fragment, MP19-25G, the first 25 amino acids of the MP19 polypeptide (Figure 4B). These data indicate that the first 25 amino acids in the MP19 polypeptide are sufficient to direct the fused EGFP molecule to the membrane and to associate within the membrane as an integral membrane protein. Under normal circumstances, expressed EGFP without the MP19 fusion protein attached is a completely soluble protein within the cytoplasm (Figure 5A).
Computer analysis of the MP19 amino acid sequence using several different prediction servers (TMHMM-2.0 - Prediction of transmembrane helices in proteins; SignalP - Presence and location of signal peptide cleavage sites; SIGFIND - Signal peptide prediction server; and TargetP - Prediction of subcellular location and cleavage site) [24-28] indicated that MP19 did contain a signal peptide (with a probability of 99%) within the first 28 amino acids of MP19 (Table 1), and served as a signal anchor within the first 25 amino acids. Analysis of the peptide for signal attributes was carried out following truncation of the peptide by one amino acid from the COOH-terminal end. As the amino acids were deleted one-by-one, the signal anchor and peptide changed to only a signal anchor at amino acid 25, then ceased to have any signal activity from amino acid 20 on down (Table 1). Therefore, MP19-25 would likely contain the complete membrane target signal. These prediction servers, while being very powerful, only predict the possible function of amino acid sequences. These data do not prove that these initial amino acids in the MP19 molecule make up both a signal peptide and a signal anchor; they only indicate the probability of one or the other. Clearly, MP19-25 with EGFP fused to the COOH-terminus of the truncated peptide (MP19-25G) changed the EGFP molecule from a totally soluble, cytoplasmic protein (Figure 5A) to a protein that associated with the cell membrane as an integral membrane protein (Figure 4B, Alkaline pellet), and with lipid rafts (Figure 4B, Triton pellet).
Since this MP19-25/EGFP molecule (MP19-25G) clearly became an intrinsic membrane protein, the first 25 amino acids of MP19 must itself integrate into the cell membrane (shown schematically in Figure 6A). The same results are observed with the peptide at 36 (MP19-36G; Figure 6B) or 64 (MP19-64G; Figure 6C) amino acids. However, it appears that the NH2-terminal end of MP19-25 does not completely span the membrane and protrude into the extracellular space, due to the fact that when we constructed an expression vector that contained EGFP fused to the NH2-terminal end of MP19-25 (GMP19-25) rather than the COOH-terminal end (MP19-25G), the fusion protein became completely soluble in the cytoplasm (Figure 5B,C), as is seen with expressed EGFP alone (Figure 5A). The above result was also observed when EGFP was fused to the NH2-terminal end of MP19-36 (GMP19-36; Figure 5D,E), thus indicating that the length of the MP19 peptide was not affecting the insertion of MP19-25 into the membrane. Figure 7A,B demonstrates a representation of the the results one would observe if the NH2-terminal end of MP19 projected into the extracellular space (Figure 7A) or if EGFP were able to integrate into the cell membrane while fused to MP19-25 (Figure 7B). Neither of these possibilities appears to be possible from our data, rather, the fusion protein remains free in the cytoplasm, as indicated in Figure 7C. Another possible interpretation of these data would involve the loss of signal properties in the first 25 amino acids of the MP19 molecule when they are fused to the COOH-terminus of EGFP. However, when intact MP19 is fused to the COOH-terminal end of EGFP (GMP19), the fusion protein transported to the cell membrane (Figure 5F,G), exactly as seen when MP19 was fused to the NH2-terminal end of EGFP (MP19G; Figure 3A,B). Western blot analysis of GMP19 and GMP19-25 also showed that the intact MP19 molecule with EGFP fused to the NH2-terminal end (GMP19) was associated with the Triton-insoluble fraction (lipid rafts; Figure 8A) while MP19-25 with EGFP fused to the NH2-terminal end (GMP19-25) was totally soluble, indicating that it did not associate with the cell membrane (Figure 8B). This result does not indicate that the first 25 amino acids of the MP19 molecule are now inserting into the cell membrane, it does indicate that the first 25 amino acids continue to serve as a membrane signal (since the protein became an integral membrane protein) and the other transmembrane segments contribute to the integration into the cell membrane, as shown shematically in Figure 9.
In summary, these data indicate that: (1) the first 24 amino acids of MP19 are a signal for transport of MP19 to the cell membrane and to carry out the initial integration into the cell membrane; (2) The NH2-terminus of the MP19 molecule integrates into the cell membrane, but the NH2-terminal end of the molecule does not span the membrane; and (3) The anchor peptide (amino acids 1-21) are not necessary for integration of the intact MP19 molecule into the cell membrane. The other transmembrane loops within the MP19 molecule are sufficient to achieve stable integration into the cell membrane. Figure 10 shows a summary of the data, with identification of each construct used and the result of expression of the fusion protein products. The above results now require a redesign of the topology of MP19 in the cell membrane since our data do not support previous conjectures about the topology of wildtype MP19 [2,4,14]. Figure 11 shows the possible arrangement of MP19 in the cell membrane, with the first 21 amino acids inserted into the membrane as a type of "anchor" peptide, thus allowing the rest of the MP19 molecule to properly insert into the cell membrane. Further investigations should shed light on the overall topology of the intact MP19 molecule.
This research was supported in part by National Institutes of Health grants R01 EY11516 and R01 EY12301 to RLC, and T32 EY07092, P30 EY06360, C06 EY06307, the Knights Templar Educational Foundation of Georgia, Inc., and a Departmental Grant from Research to Prevent Blindness, Inc. Some of these data were presented in abstract form at the 2002 meeting of the Association for Research in Vision and Ophthalmology.
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