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MP20)
Experimental Eye Research 103 (2012) 115e116
Contents lists available at SciVerse ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Focus on Molecules: Lens intrinsic membrane protein (LIM2/MP20) Geoffrey J. Maher a, *, Graeme C. Black a, b, Forbes D. Manson a a
School of Biomedicine, The University of Manchester, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9PT, UK b Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9WL, UK
a r t i c l e i n f o Article history: Available online 16 August 2011 Keywords: lens cataract membrane syncytium adhesion
1. Structure The lens intrinsic membrane protein 2 (LIM2) gene encodes the conserved 173 amino acid integral membrane protein MP20 (also known as MP19; NP_001155220). The original hydropathy analysis of MP20 predicted four transmembrane domains, short intracellular N- and C-termini, two extracellular loops, the first being larger than the second, and a short intracellular loop. Initial experiments confirmed the C-terminus was cytoplasmic but data for the N-terminus and predicted loop domains gave contradictory results. The subsequent identification of glycosylation sites in the first loop of bovine MP20 confirmed its extracellular location, supporting the overall topology of the original predicted model (Fig. 1A). This first extracellular loop of MP20 contains a 6 amino acid W-GLW-C-C motif of unknown function (Prosite motif PDOC01045). The four transmembrane topology and W-GLW-C-C motif define the PMP-22/EMP/MP20/Claudin (Pfam00822) family of proteins, a diverse group of proteins with roles in intercellular adhesion and protein trafficking. Members of this protein family include claudins, epithelial myelin proteins (EMPs), peripheral myelin protein 22 (PMP22), transmembrane protein 114 (TMEM114), TMEM235 and the voltage-dependent calcium channel gamma subunits (CACNGs). Crosslinking experiments detected multimers of Mp20 as large as hexamers and the dominant negative effect of low amounts of the To3 mutant Mp20
* Corresponding author. E-mail address: [email protected] (G.J. Maher). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.08.006
(see Section 3) supports the multimer organisation of the protein. In the presence of native lens lipids, ovine Mp20 forms octomers (Fig. 1B), and possibly larger multimers, composed of tetrameric subunits (Gonen et al., 2008). 2. Function MP20 is exclusively expressed in the lens fibre cells where it is the second most prevalent membrane protein after MIP/AQP0. The protein displays differential localization during fibre cell differentiation: in peripheral nucleated cells Mp20 is cytoplasmic, but in deeper fibre cells it is localized at the plasma membrane. The relocalization of Mp20 coincides with the loss of fibre cell nuclei and correlates with the formation of a diffusion barrier caused by a decrease in the extracellular space (Grey et al., 2003) suggesting a role for Mp20 in cellecell adhesion. Although their lenses are grossly normal, the adhesion between lens fibre cells from Mp20 deficient (Lim2Gt/Gt) mice is less than that of lenses from wildtype mice. Further evidence supporting a role for MP20 in cellecell adhesion is its interaction with galectin-3, a modulator of cell-tocell adhesion which undergoes a similar redistribution during lens differentiation. MP20 also has homology to PMP22 and the claudins which localize at tight junctions and modulate the paracellular movement of small molecules between epithelial cells. Mp20 is required for the formation of syncytia between lens fibre cells, a structure of cellecell fusions which facilitates the movement of large molecules between cells. Bovine Mp20 co-localizes with the gap junction protein connexin 46 (Cx46) in the lens cortex and the lens core of Lim2Gt/Gt mice have reduced expression of Cx46, although the loss of Mp20 has no effect on Cx46 expression in the cortex (Shi et al., 2011). Fibre cells in the lens cortex and core of Lim2Gt/Gt mice have reduced coupling conductance and slight membrane depolarization, suggesting that Mp20 contributes to the electrophysiological properties of the lens. Expression of a number of cytoskeletal proteins is reduced in the lens core of Lim2Gt/Gt mice due to proteolytic degradation (Shi et al., 2011). A possible role of MP20 in cell signalling is suggested by its C-terminal phosphorylation and its interaction with calmodulin. Mp20 is associated with transparency of the lens (see Section 3) and thus Mp20 may have a direct role in maintaining transparency by reducing light scatter through its role in intercellular adhesion. Mp20 may also play indirect roles through maintaining the integrity of the lens cytoskeleton, maintaining
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wildtype littermates and their lens fibres display an irregular arrangement. Homozygous p.C51R mice (also known as Aca47 mice) are more severely affected, having smaller lenses with nuclear cataracts. Subsequent to its association with an ocular phenotype in mice, mutations in LIM2 were identified in recessive forms of human congenital and pre-senile cataract. A homozygous mutation, p.F105V, in the third transmembrane domain is associated with mild pulverulent cataract with a pre-senile or senile onset (20e70 years). The p.G154E mutation is predicted to disrupt the fourth transmembrane domain and is associated with recessive congenital or infantile total cataracts. Affected individuals also had nystagmus and dense amblyopia. Screening of 145 age-related cataract patients failed to identify a convincing association with mutations in LIM2. Fig. 1. A) Predicted topology of MP20. The six amino acid W-GLW-C-C motif present in the first extracellular loop is coloured orange. Glycosylated tryptophan residues (W43 and W61) are labelled with a solid blue marker. A predicted, but experimentally unconfirmed, N-glycosylation site is present at p.N62 (outlined blue marker). Two phosphorylation sites (S170 and T171) (purple) are present in the intracellular C-terminus. Amino acids mutated in human forms of recessive cataract are coloured red. Mutated amino acids in mouse models are outlined in red. The four exons of MP20 are delimited by red lines. B) Three-dimensional reconstructions of negatively stained oligomeric ovine Mp20enative lipid complexes, as produced by Gonen et al., 2008. Predicted oligomers of tetramers of Mp20 forming (i) the six-bladed windmill particle, (ii) the rectangular particle, (iii) the triangular particle, and (iv) the octameric particle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
intercellular communication, or through the creation a uniform refractive index by allowing the even distribution of protein in the lens fibres (via the lens syncytium). 3. Disease involvement A Lim2 homozygous missense mutation, p.G15V, was identified in the To3 (total opacity of lens #3) mouse, an ENU-generated mouse which displays autosomal semi-dominant congenital cataracts (Steele et al., 1997). Heterozygous mice have dense cataracts whilst homozygous mutant mice have microphthalmia, rupturing of the lens, and disorganised primary and secondary fibres, some of which still retain their nuclei. The mutated residue is located in the first transmembrane domain (Fig. 1A) and in vitro the mutant Mp20 fails to insert in the plasma membrane, possibly remaining in the ER-Golgi intermediate compartment. To confirm the pathogenicity of the p.G15V mutation, Lim2To3 transgenic mice were generated. Lim2To3 mice display a similar phenotype to the ENU-generated To3 mice, although the heterozygotes are more severely affected, displaying a reduction in lens size. The Lim2To3 phenotype was observed even though the transgenic mutant Lim2 allele was expressed at lower levels than the wildtype allele, suggesting the mutation acts in a dominant negative manner. Further support for the dominant negative nature of the mutation is that Mp20 deficient mice have a less severe phenotype than the To3 mice. Mice heterozygous for a mutation in the second cysteine of the W-GLW-C-C motif (p.C51R) have smaller lenses that their
4. Future studies The evidence to date suggests a role for MP20 in intercellular adhesion and cellecell fusion, although its precise role has yet to be established. How MP20 translocates to the membrane has yet to be determined, as does any potential role of intracellular MP20 in the early differentiating fibres. The functional consequences of mutating the cysteine in the W-GLW-C-C motif have to be determined and such studies may also be relevant to other members of the Pfam00822 family. The motif may play a role in the adhesion process, as is predicted for the claudins and other family members. It is also unknown whether MP20 interacts with other members of the Pfam00822 family and whether the interaction with galectin-3 is involved in cellecell adhesion or cell signalling. Identification of interaction partners may also reveal the role of MP20 in the formation of the lens syncytium. It remains to be determined if increased protein degradation and intercellular resistance are directly or indirectly due to loss of Mp20. The p.G154E mutation associated with human congenital cataract is located immediately downstream of the splice acceptor site of intron 4 raising the possibility that this mutation alters the pre-mRNA splicing of Lim2. Lastly, the presence of microphthalmia in the homozygous To3 and Aca47 mice suggests that LIM2 may represent a good candidate gene for recessive cases of microphthalmia in humans. Acknowledgements GJM is supported by Fight for Sight (Ref: 1658).
References Gonen, T., Hite, R.K., Cheng, Y., Petre, B.M., Kistler, J., Walz, T., 2008. Polymorphic assemblies and crystalline arrays of lens tetraspanin MP20. J. Mol. Biol. 376, 380e392. Grey, A.C., Jacobs, M.D., Gonen, T., Kistler, J., Donaldson, P.J., 2003. Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier. Exp. Eye Res. 77, 567e574. Shi, Y., De Maria, A.B., Wang, H., Mathias, R.T., FitzGerald, P.G., Bassnett, S., 2011. Further analysis of the lens phenotype in Lim2-deficient mice. Invest. Ophthalmol. Vis. Sci. Jul 20. Epub ahead of print. Steele Jr., E.C., Kerscher, S., Lyon, M.F., Glenister, P.H., Favor, J., Wang, J., Church, R.L., 1997. Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3. Mol. Vis. 3, 5.
Contents lists available at SciVerse ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Focus on Molecules: Lens intrinsic membrane protein (LIM2/MP20) Geoffrey J. Maher a, *, Graeme C. Black a, b, Forbes D. Manson a a
School of Biomedicine, The University of Manchester, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9PT, UK b Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9WL, UK
a r t i c l e i n f o Article history: Available online 16 August 2011 Keywords: lens cataract membrane syncytium adhesion
1. Structure The lens intrinsic membrane protein 2 (LIM2) gene encodes the conserved 173 amino acid integral membrane protein MP20 (also known as MP19; NP_001155220). The original hydropathy analysis of MP20 predicted four transmembrane domains, short intracellular N- and C-termini, two extracellular loops, the first being larger than the second, and a short intracellular loop. Initial experiments confirmed the C-terminus was cytoplasmic but data for the N-terminus and predicted loop domains gave contradictory results. The subsequent identification of glycosylation sites in the first loop of bovine MP20 confirmed its extracellular location, supporting the overall topology of the original predicted model (Fig. 1A). This first extracellular loop of MP20 contains a 6 amino acid W-GLW-C-C motif of unknown function (Prosite motif PDOC01045). The four transmembrane topology and W-GLW-C-C motif define the PMP-22/EMP/MP20/Claudin (Pfam00822) family of proteins, a diverse group of proteins with roles in intercellular adhesion and protein trafficking. Members of this protein family include claudins, epithelial myelin proteins (EMPs), peripheral myelin protein 22 (PMP22), transmembrane protein 114 (TMEM114), TMEM235 and the voltage-dependent calcium channel gamma subunits (CACNGs). Crosslinking experiments detected multimers of Mp20 as large as hexamers and the dominant negative effect of low amounts of the To3 mutant Mp20
* Corresponding author. E-mail address: [email protected] (G.J. Maher). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.08.006
(see Section 3) supports the multimer organisation of the protein. In the presence of native lens lipids, ovine Mp20 forms octomers (Fig. 1B), and possibly larger multimers, composed of tetrameric subunits (Gonen et al., 2008). 2. Function MP20 is exclusively expressed in the lens fibre cells where it is the second most prevalent membrane protein after MIP/AQP0. The protein displays differential localization during fibre cell differentiation: in peripheral nucleated cells Mp20 is cytoplasmic, but in deeper fibre cells it is localized at the plasma membrane. The relocalization of Mp20 coincides with the loss of fibre cell nuclei and correlates with the formation of a diffusion barrier caused by a decrease in the extracellular space (Grey et al., 2003) suggesting a role for Mp20 in cellecell adhesion. Although their lenses are grossly normal, the adhesion between lens fibre cells from Mp20 deficient (Lim2Gt/Gt) mice is less than that of lenses from wildtype mice. Further evidence supporting a role for MP20 in cellecell adhesion is its interaction with galectin-3, a modulator of cell-tocell adhesion which undergoes a similar redistribution during lens differentiation. MP20 also has homology to PMP22 and the claudins which localize at tight junctions and modulate the paracellular movement of small molecules between epithelial cells. Mp20 is required for the formation of syncytia between lens fibre cells, a structure of cellecell fusions which facilitates the movement of large molecules between cells. Bovine Mp20 co-localizes with the gap junction protein connexin 46 (Cx46) in the lens cortex and the lens core of Lim2Gt/Gt mice have reduced expression of Cx46, although the loss of Mp20 has no effect on Cx46 expression in the cortex (Shi et al., 2011). Fibre cells in the lens cortex and core of Lim2Gt/Gt mice have reduced coupling conductance and slight membrane depolarization, suggesting that Mp20 contributes to the electrophysiological properties of the lens. Expression of a number of cytoskeletal proteins is reduced in the lens core of Lim2Gt/Gt mice due to proteolytic degradation (Shi et al., 2011). A possible role of MP20 in cell signalling is suggested by its C-terminal phosphorylation and its interaction with calmodulin. Mp20 is associated with transparency of the lens (see Section 3) and thus Mp20 may have a direct role in maintaining transparency by reducing light scatter through its role in intercellular adhesion. Mp20 may also play indirect roles through maintaining the integrity of the lens cytoskeleton, maintaining
116
G.J. Maher et al. / Experimental Eye Research 103 (2012) 115e116
wildtype littermates and their lens fibres display an irregular arrangement. Homozygous p.C51R mice (also known as Aca47 mice) are more severely affected, having smaller lenses with nuclear cataracts. Subsequent to its association with an ocular phenotype in mice, mutations in LIM2 were identified in recessive forms of human congenital and pre-senile cataract. A homozygous mutation, p.F105V, in the third transmembrane domain is associated with mild pulverulent cataract with a pre-senile or senile onset (20e70 years). The p.G154E mutation is predicted to disrupt the fourth transmembrane domain and is associated with recessive congenital or infantile total cataracts. Affected individuals also had nystagmus and dense amblyopia. Screening of 145 age-related cataract patients failed to identify a convincing association with mutations in LIM2. Fig. 1. A) Predicted topology of MP20. The six amino acid W-GLW-C-C motif present in the first extracellular loop is coloured orange. Glycosylated tryptophan residues (W43 and W61) are labelled with a solid blue marker. A predicted, but experimentally unconfirmed, N-glycosylation site is present at p.N62 (outlined blue marker). Two phosphorylation sites (S170 and T171) (purple) are present in the intracellular C-terminus. Amino acids mutated in human forms of recessive cataract are coloured red. Mutated amino acids in mouse models are outlined in red. The four exons of MP20 are delimited by red lines. B) Three-dimensional reconstructions of negatively stained oligomeric ovine Mp20enative lipid complexes, as produced by Gonen et al., 2008. Predicted oligomers of tetramers of Mp20 forming (i) the six-bladed windmill particle, (ii) the rectangular particle, (iii) the triangular particle, and (iv) the octameric particle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
intercellular communication, or through the creation a uniform refractive index by allowing the even distribution of protein in the lens fibres (via the lens syncytium). 3. Disease involvement A Lim2 homozygous missense mutation, p.G15V, was identified in the To3 (total opacity of lens #3) mouse, an ENU-generated mouse which displays autosomal semi-dominant congenital cataracts (Steele et al., 1997). Heterozygous mice have dense cataracts whilst homozygous mutant mice have microphthalmia, rupturing of the lens, and disorganised primary and secondary fibres, some of which still retain their nuclei. The mutated residue is located in the first transmembrane domain (Fig. 1A) and in vitro the mutant Mp20 fails to insert in the plasma membrane, possibly remaining in the ER-Golgi intermediate compartment. To confirm the pathogenicity of the p.G15V mutation, Lim2To3 transgenic mice were generated. Lim2To3 mice display a similar phenotype to the ENU-generated To3 mice, although the heterozygotes are more severely affected, displaying a reduction in lens size. The Lim2To3 phenotype was observed even though the transgenic mutant Lim2 allele was expressed at lower levels than the wildtype allele, suggesting the mutation acts in a dominant negative manner. Further support for the dominant negative nature of the mutation is that Mp20 deficient mice have a less severe phenotype than the To3 mice. Mice heterozygous for a mutation in the second cysteine of the W-GLW-C-C motif (p.C51R) have smaller lenses that their
4. Future studies The evidence to date suggests a role for MP20 in intercellular adhesion and cellecell fusion, although its precise role has yet to be established. How MP20 translocates to the membrane has yet to be determined, as does any potential role of intracellular MP20 in the early differentiating fibres. The functional consequences of mutating the cysteine in the W-GLW-C-C motif have to be determined and such studies may also be relevant to other members of the Pfam00822 family. The motif may play a role in the adhesion process, as is predicted for the claudins and other family members. It is also unknown whether MP20 interacts with other members of the Pfam00822 family and whether the interaction with galectin-3 is involved in cellecell adhesion or cell signalling. Identification of interaction partners may also reveal the role of MP20 in the formation of the lens syncytium. It remains to be determined if increased protein degradation and intercellular resistance are directly or indirectly due to loss of Mp20. The p.G154E mutation associated with human congenital cataract is located immediately downstream of the splice acceptor site of intron 4 raising the possibility that this mutation alters the pre-mRNA splicing of Lim2. Lastly, the presence of microphthalmia in the homozygous To3 and Aca47 mice suggests that LIM2 may represent a good candidate gene for recessive cases of microphthalmia in humans. Acknowledgements GJM is supported by Fight for Sight (Ref: 1658).
References Gonen, T., Hite, R.K., Cheng, Y., Petre, B.M., Kistler, J., Walz, T., 2008. Polymorphic assemblies and crystalline arrays of lens tetraspanin MP20. J. Mol. Biol. 376, 380e392. Grey, A.C., Jacobs, M.D., Gonen, T., Kistler, J., Donaldson, P.J., 2003. Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier. Exp. Eye Res. 77, 567e574. Shi, Y., De Maria, A.B., Wang, H., Mathias, R.T., FitzGerald, P.G., Bassnett, S., 2011. Further analysis of the lens phenotype in Lim2-deficient mice. Invest. Ophthalmol. Vis. Sci. Jul 20. Epub ahead of print. Steele Jr., E.C., Kerscher, S., Lyon, M.F., Glenister, P.H., Favor, J., Wang, J., Church, R.L., 1997. Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3. Mol. Vis. 3, 5.