Human Molecular Genetics Advance Access originally published online on May 4, 2006
Human Molecular Genetics 2006 15(12):1972-1983; doi:10.1093/hmg/ddl120
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Nance–Horan syndrome protein, NHS, associates with epithelial cell junctions
1Department of Ophthalmology, Flinders University, Bedford Park, SA 5042, Australia, 2Save Sight Institute, Sydney Eye Hospital, University of Sydney, Sydney 2001, Australia, 3Women's and Children's Hospital, North Adelaide, SA 5006, Australia, 4Centre for Eye Research Australia, Royal Victorian Ear and Eye Hospital, University of Melbourne, East Melbourne, Victoria 3002, Australia and 5Department of Paediatrics, University of Adelaide, Australia
* To whom correspondence should be addressed. Tel: +61 0882045892; Fax: +61 0882770899; Email: shiwani.sharma{at}flinders.edu.au
Received March 12, 2006; Accepted April 29, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nance–Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance–Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance–Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nance–Horan syndrome is an X-linked genetic disorder involving congenital cataracts, dental anomalies and in some cases mental retardation and behavioural problems (1
–3). Affected males have severe bilateral cataracts leading to profound vision loss and requiring early surgery. Microcornea and microphthalmia have also been reported in some families (4–6). Dental abnormalities include screwdriver-shaped incisors, supernumerary maxillary incisors and diastema (5,6). Heterozygous females display similar but less severe features than affected males (5,7). The minimal region for the syndrome was mapped using genetic analysis (8,9). We, and subsequently others, identified causative mutations in the novel gene, NHS (Nance–Horan syndrome) with unknown function, in affected families (10–12). Frame-shift or nonsense mutations lead to premature truncation of the protein. In mouse, Nhs is expressed in developing brain, eye and teeth primordia in a spatially and temporally regulated manner consistent with developmental defects found in these organs in individuals with the Nance–Horan syndrome (10).
The Xcat mutant mouse identified in a cataract mutation screen exhibits X-linked congenital cataract with males expressing bilateral total lens opacity and heterozygous females displaying phenotypic variation, from totally clear to totally opaque lens (13). Phenotypic similarity accompanied by genetic evidence suggested that Xcat mouse is the animal model for the Nance–Horan syndrome (13,14). Recently, a large insertion mutation in the first intron of Nhs/Nhs1 gene was identified as the cause of congenital cataract in the Xcat mouse, which confirms the latter as an animal model of the Nance–Horan syndrome (15). Two variants of the Nhs gene, Nhs1 and Nhs_v1, are expressed in developing mouse embryo as a result of transcription from two start sites in exons 1 and 1A, respectively, and alternative splicing of exon 2. In the Xcat mouse, insertion inhibits the expression of Nhs1, initiated in exon 1 of Nhs, and results in exclusive expression of Nhs_v1 (15). Nhs protein was detected in lens fibre cells in newborn wild-type animals but not in the Xcat mouse; thus, Huang et al. (15) proposed this difference to underlie the mechanism of cataract formation in the latter.
Previously, we described two major transcripts of the human NHS gene with different transcription start sites in exons 1 and 1b, respectively, and predicted to encode two protein isoforms, NHS-A (same as mouse Nhs1) and NHS-B (different from mouse Nhs_v1), respectively, 1630 and 1335 amino acids long (10). The N-terminus of NHS-A precedes the initiation codon of NHS-B. Consistent with the finding in mouse (15), the present work details the expression of the third isoform NHS-1A (same as mouse Nhs_v1) with transcription and translation start site in exon 1A, downstream of exon 1 (Fig. 1). This isoform differs from NHS-A at the N-terminus and is predicted to encode a 1474 amino acid protein. We demonstrate for the first time that the NHS-A and NHS-1A isoforms of NHS are differentially expressed and distinctly targeted in mammalian cells under normal circumstances. We show that the NHS-A isoform is targeted to the cell periphery and/or cytoplasm in a cell-type-dependent manner, whereas the NHS-1A isoform is a cytoplasmic protein. Expression of multiple NHS variants and their localization to distinct cellular compartments suggest complex regulation and multiple functions of the gene. We also report detailed analysis of Nhs expression in the developing mouse lens and demonstrate transcript and protein expression in the lens epithelium and not in fibre cells. Additionally, we provide evidence that in the lens epithelium the NHS-A isoform is localized at cell–cell contact sites, which suggests that intercellular contacts likely underlie cataractogenesis in the Nance–Horan syndrome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of complete cDNA and NHS-specific antibody
The 1368-nucleotide 5' coding region of the NHS cDNA amplified from SRA 01/04 human lens epithelium cDNA and the 3834-nucleotide overlapping 3' coding cDNA including the termination codon amplified from human hippocampal cDNA were used to create the complete coding cDNA (see Materials and Methods). Cloning and sequencing of the 5' cDNA amplified from SRA 01/04 cells revealed an additional 66 bp exon, 3a, between the previously reported exons 3 and 1b (10
) (Fig. 1). This extends the existing 1630 amino acid NHS-A open-reading frame by 22 amino acids. The exon 3a sequence was absent in one of the six clones sequenced, thereby suggesting its alternate splicing. The presence of exon 3a sequence in the human NHS mRNA AY456992
[GenBank]
and AY456993
[GenBank]
in the GenBank supports our finding. Screening this exon in individuals affected with the Nance–Horan syndrome did not reveal any mutation when compared with normal controls (Burdon K. and Sharma S., unpublished data).
Polyclonal anti-NHS antibody against the C-terminal peptide of NHS protein was affinity purified from immune rabbit serum. The specificity of the antibody was determined by its ability to detect transiently expressed NHS protein. The complete NHS-A isoform and the C-terminal 1278 amino acids (375–1652) encoded from exons 5 to 8 and represented in all NHS isoforms (Fig. 1) were transiently expressed as green fluorescent protein (GFP) fusions in human embryonic kidney (HEK) 293A cells. Upon western blotting, the affinity-purified antibody, respectively, detected >250 and 
250 kDa specific proteins corresponding to GFP–NHS-A and GFP–NHS(375–1652) (Fig. 2). The sizes of both the fusion proteins were significantly greater than the expected sizes of 208 and 167 kDa, respectively. The NHS protein is predicted not to have a well-defined secondary conformation that can cause slower migration of the protein in SDS–PAGE. The largely unfolded Gir2 protein of Saccharomyces cerevisiae exhibits similar anomalous behaviour in SDS–PAGE (16).
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Differential expression of NHS isoforms
In our previous study, in situ hybridization of mouse embryo sections indicated some spatial differences in expression of the Nhs isoforms (10
). To further investigate this preliminary observation, expression of NHS was determined in cultured mammalian cells. SRA 01/04 and 
TN4 cell lines, respectively, derived from human and mouse lens epithelium (17,18) and HEK 293A human and NIH 3T3 mouse fibroblast cell lines were used for this study. SRA 01/04 and TN4 cells grow as monolayer in culture and express the lens protein A crystallin (17,18). RT–PCR with NHS-A-specific primers designed to amplify a region from exon 1 to exon 4 revealed an amplicon of 542 bp in SRA 01/04 and TN4 cells (Fig. 3A). No amplification was detected in HEK 293A and NIH 3T3 cells with this primer pair. However, RT–PCR with gene-specific primers designed to amplify a region from exon 6 to exon 8, common to all the three isoforms, resulted in a 206 bp amplicon in human and mouse lens epithelium and fibroblast cells suggesting that HEK 293A and NIH 3T3 fibroblast cells express NHS but not the NHS-A isoform. 5'-RACE of NHS in HEK 293A cells revealed expression of the NHS-1A isoform.
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Western analysis of endogenous NHS in these cells with the anti-NHS-specific antibody detected a single >250 kDa protein corresponding to NHS-A in SRA 01/04 and
TN4 cells and a major 250 kDa protein corresponding to NHS-1A in HEK 293A and NIH 3T3 cells (Fig. 3B). Once again, these protein sizes are larger than those predicted for the human and mouse NHS-A and NHS-1A (Table 1), which is in agreement with slower migration of ectopically expressed NHS proteins observed in SDS–PAGE (Fig. 2). The smaller <150 kDa protein band present in HEK 293A and NIH 3T3 cells is probably due to partial degradation of the protein (Fig. 3B). However, the possibility that it represents a non-specific protein or another NHS isoform cannot be excluded. Furthermore, the >250 kDa protein was also detected in MDCK (Madin–Darby canine kidney) epithelium and SH-SY5Y human neuroblastoma cells suggesting expression of NHS-A isoform in these cell lines (Fig. 3B). The RT–PCR and western blotting data together provide evidence for differential expression of NHS-A and -1A isoforms in mammalian cells.
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Localization of NHS isoforms to distinct subcellular compartments
To study subcellular distribution of the two NHS isoforms, endogenous protein in MDCK, SH-SY5Y and NIH 3T3 cells was immunolabelled with the anti-NHS antibody. MDCK cells form into a polarized epithelium by developing intercellular junctions in confluent culture (19
,20). The NHS-A isoform expressed in these cells was primarily localized at the cell periphery, although some cytoplasmic localization was also observed (Fig. 4A). The protein was distributed in a punctate fashion at the cell periphery with the signal being more intense at the sites of contact between three adjoining cells (Fig. 4B). The Z-serial optical sections through the cells revealed the protein at the apical membrane both along the X–Y plane and the Z-axis (Fig. 4C). Cytoplasmic distribution of the protein was more obvious in these optical sections. In SH-SY5Y cells that grow as neuroblastic clusters with small neurites (21), NHS-A was found in the cytoplasm but not at the cell periphery (Fig. 4D). The NHS-1A isoform expressed in NIH 3T3 cells localized mainly in the cytoplasm (Fig. 4E). Similar distribution of the protein was observed in HEK 293A cells (data not shown).
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Targeting of NHS-A in mammalian cells
To further understand the targeting of NHS-A in different types of cells, we ectopically expressed the full-length NHS-A fused to GFP, in epithelial and fibroblast cells. The GFP–NHS-A fusion protein detectable with the anti-NHS antibody (Fig. 2) was transiently expressed in MDCK, SRA 01/04 and HEK 293A cells. Confocal microscopy of transfected cells revealed localization of GFP–NHS-A at the cell periphery and in the cytoplasm in MDCK cells (Fig. 5A). However, in both SRA 01/04 and HEK 293A cells, it localized in the cytoplasm. GFP was distributed throughout the cell in each cell type upon transfection with control vector. Thus, targeting of NHS-A to the cell periphery seems to be cell-type-dependent.
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To determine the region of NHS-A responsible for targeting the protein to cell periphery in MDCK cells, the N- and C-terminal portions of the protein were ectopically expressed in MDCK, SRA 01/04 and HEK 293A cells. The N-terminal 2–442 amino acids of NHS-A encoded from exon 1 to the start of exon 6 (with and without exon 3a) were fused downstream of GFP to create the GFP–NHSA442 fusion constructs. Upon transient transfection of HEK 293A cells, these constructs expressed fusion proteins of the expected sizes that could be detected with the anti-GFP antibody (Fig. 6). The C-terminal 1278 amino acids encoded from exons 5 to 8 were included in GFP–NHS(375–1652) fusion construct (Fig. 2). The GFP–NHSA442 and GFP–NHS(375–1652) share a 68 amino acid overlap. In MDCK cells transfected with either GFP–NHSA442 construct, the fusion protein primarily localized to the cell periphery, although cytoplasmic distribution was also noted (Fig. 5B) (data not shown). Upon expression in SRA 01/04 and HEK 293A cells, this fusion protein localized in the cytoplasm and often formed aggregates (Fig. 5B). The full-length GFP–NHS-A did not form such aggregates, which may be an artefact due to over expression of the partial GFP–NHSA442 protein. Ectopically expressed GFP–NHS(375–1652) fusion protein localized in the cytoplasm but never at the cell periphery in MDCK, SRA 01/04 and HEK 293A cells (Fig. 5B). These data suggest that the first 374 residues of NHS-A are responsible for targeting the protein to the cell periphery in MDCK cells.
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Tissue expression of NHS
To determine which isoform is expressed in eye and brain, the two organs affected in the Nance–Horan syndrome, RT–PCR was performed on RNA isolated from human lens, retina and brain tissues. The NHS-A-specific primers designed to amplify across exon 3a resulted in an amplicon of
540 bp in lens and retina and one of 475 bp in fetal and adult brain (Fig. 7A). These two products correspond to two NHS-A mRNA isoforms, which differ in the inclusion of exon 3a (discussed earlier). Sequencing confirmed the presence of exon 3a in the 540 bp amplicon from the lens and its absence in the 475 bp amplicons from fetal and adult brain. Hence, both eye and brain express the NHS-A isoform, and exon 3a is alternately spliced in these tissues. The expression of NHS-A in lens and brain correlates well with its expression in the lens epithelium and neuroblastoma cell lines derived from these tissues. Western blotting with the anti-NHS antibody detected a single >250 kDa protein in adult human and mouse lens, which corresponds to NHS-A and indicates exclusive expression of this isoform in the lens tissue (Fig. 7B).
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Expression in developing ocular lens
As mutations in the NHS gene cause congenital cataracts, the NHS protein function is clearly essential for normal lens development. To further understand its role, we performed detailed analysis of Nhs expression in developing mouse lens. In situ hybridization of sections from developing lens with riboprobe generated from a region in exon 6 of Nhs revealed expression in invaginating lens placode at embryonic day (E) 10.5, lens vesicle at E11.5 and in optic cup at these two stages of development (Fig. 8). At E12.5, when the posterior lens vesicle cells differentiate into primary lens fibre cells and elongate to fill the cavity of the vesicle (22
), expression was observed both in anterior lens vesicle cells and in primary fibre cells. Thereafter from E14.5 to E16.5 and E18.5, Nhs expression became progressively restricted to the anterior lens epithelium. Nhs expression was also downregulated upon differentiation of epithelial cells into fibre cells at the lens equator. Expression in the anterior lens epithelium continued after birth and in adult lens (Fig. 9). No Nhs expression was detected in the lens fibre cells of postnatal lens, and downregulation of expression in the lens epithelium was evident in the transition zone below the lens equator, where after cell division epithelial cells elongate and differentiate into fibre cells (22).
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Immunolabelling of rat lens sections with the anti-NHS antibody revealed protein in the anterior lens epithelium in postnatal day (P) 1 and P11 lenses (Fig. 10A). The NHS protein was downregulated in the elongating fibre cells in the transition zone at the lens equator and no protein was detectable in mature fibre cells. This protein distribution in the rat lens is consistent with presence of Nhs mRNA in the anterior lens epithelium of both pre- and post- natal mouse lens. Immunolabelling of whole central lens epithelium from P10 rat showed Nhs localized at the cell periphery in apical aspect and in the cytoplasm in basal aspect of the epithelium (Fig. 10B). Similar peripheral localization of NHS was observed in whole central epithelium from postmortem human lens (data not shown). Localization of NHS at the cell periphery in the lens epithelium in vivo is in accordance with the distribution of NHS-A in cultured MDCK epithelium cells (Fig. 4).
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Co-localization of NHS-A with tight junctions
Endogenous NHS-A and exogenous GFP–NHS-A fusion protein localized at the cell boundary in MDCK cells alone. This localization is inferred to be associated with intercellular contacts established by these cells during their polarization in culture (19
,20). Because NHS-A localizes to the apical membrane in MDCK cells and lens epithelium in vivo, and tight junctions (TJs) are also located at apical membrane in polarized epithelium (23), we sought to determine whether NHS-A localizes to TJs in MDCK cells. In the Z-serial optical sections through the cells double labelled for NHS and the TJ protein ZO-1, NHS-A localized along the cell periphery at the apical membrane (Fig. 11, optical sections 1–3), as noted before, to discrete foci at the lateral membranes (Fig. 11, optical sections 4–5) and to the cytoplasm in the basal part of cells (Fig. 11, optical section 6). ZO-1 was regularly distributed at the cell membrane in the optical sections of the apical region (Fig. 11, optical sections 1–3) where it co-localized with NHS-A (Fig. 11, optical sections 1–3, merge) but was absent in the optical sections of the basal region (Fig. 11, optical sections 5–6). Discrete foci of NHS-A at the lateral membrane did not co-localize with ZO-1 (Fig. 11, optical sections 4–5, merge). We also determined the presence of NHS-A at adherens junctions located basal to TJs in epithelia. In co-localization experiments with the adherens junction protein E-cadherin present at basolateral membrane of epithelia, NHS-A did not co-localize with E-cadherin in MDCK cells (Fig. 12).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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NHS is ubiquitously expressed in human and mouse tissues (10
). It is becoming clear that several isoforms of the gene are present in mammalian species. Data presented in this study reveal that two of the isoforms encoded by NHS are differentially expressed. Epithelial and neuronal cells express the NHS-A isoform and fibroblast cells express the NHS-1A isoform. The expression of these two isoforms in mammalian cells appears to be mutually exclusive, as their simultaneous expression was not detected in any of the cell lines and tissues tested here. The two isoforms may be transcribed by alternate promoter usage involving cell-type-specific regulatory proteins. Exon 3a in NHS-A is alternatively spliced in a tissue-dependent manner creating another variant of this isoform. Differential expression of NHS-A and -1A in cultured mammalian cells in the present study corroborates well with our earlier observation in the developing mouse embryo, where NHS-A-specific probe revealed expression in neural tissues of brain and retina, lens cell bodies, olfactory epithelium, teeth primordia and whisker follicles and a probe generated from exon 6, representing all isoforms, detected additional expression in the choroid plexus and mesenchyme of heart ventricle (10). Hence, probably during development, embryonic epithelia/neuroepithelia express NHS-A, and NHS-1A is expressed in mesenchymal cells; however, this requires further investigation. Huang et al. (15) detected homologues of both these isoforms in developing mouse eye, mouth and paw that may be expressed by more than one type of cells in these tissues. Earlier we identified the NHS-B isoform in human brain (10). Furthermore, the human NHS mRNA AY456993
[GenBank]
in the GenBank suggests a 5' extension of exon 2 introducing a previously unrecognized transcription start site and translation initiation codon in this exon. The multiple variants of NHS in human and mouse identified by us and others and their expression indicate complex transcriptional regulation of the gene.
Of the mutations identified thus far in the NHS gene, exon 1 mutations disrupt only the NHS-A isoform, whereas mutations in exons 3, 5 and 6 are likely to disrupt more than one of the isoforms (10–12). However, this difference in the effect of various mutations does not seem to correlate with the severity and/or phenotypic heterogeneity of the disorder. In the Xcat mouse, the insertion mutation only disrupts Nhs1 expression, whereas expression of the other isoforms is likely to be unaffected, which may be why these mice do not display any of the non-ocular phenotypes associated with the Nance–Horan syndrome. Similarly, the Indian family with the Q39X mutation in exon 1 mainly exhibited congenital cataracts and microcornea with mild non-ocular features (12). However, family 5 reported by us and Brooks et al. (10,11) also with an exon 1 mutation (R134fsX) displayed typical NHS features. These differences in disease severity indicate the involvement of genetic modifiers. Hence, the Xcat mouse does not completely model the typical Nance–Horan syndrome.
The NHS-A and NHS-1A isoforms characterized in this work differ in their subcellular localization. NHS-A localizes distinctly in epithelial cells with intercellular contacts than in cells without such contacts. It associates with the cell membrane in confluent MDCK cells alone, whereas in SH-SY5Y cells and in sparsely seeded MDCKs, which lack intercellular contacts, NHS-A is present in the cytoplasm (Fig. 4) (unpublished data). Furthermore, exogenous NHS-A never associated with the cell membrane in SRA 01/04 and HEK 293A cells that form cell–matrix contacts but do not have well-defined cell–cell contacts. These observations suggest the involvement of intercellular contacts in localizing NHS-A to the cell membrane in epithelia. The localization of Nhs1–GFP to the cytoplasm reported in Chinese hamster ovary (CHO) cells (15) that also lack definite intercellular contacts is in line with this hypothesis. The co-localization of NHS-A with the TJ protein ZO-1 at the apical membrane of confluent MDCK cells further provides evidence for the distribution of NHS-A with the intercellular junctions in epithelia. We show that the first 374 residues of NHS-A are responsible for targeting the protein to cell membrane in epithelial cells with intercellular junctions. However, in the absence of definite cell–cell contacts, such as in SRA 01/04 and HEK 293A cells, the GFP–NHSA442 fusion protein aggregated in the cytoplasm instead of localizing to the cell periphery. These cytoplasmic aggregates may be an artefact due to an over expression of the partial protein, as the full-length GFP–NHS-A fusion was not seen to form such aggregates in SRA 01/04 or HEK 293A cells. Punctate cytoplasmic localization of partial mouse Nhs1–GFP reported in CHO cells (15) can be explained by similar reasoning and may not indicate biologically relevant distribution of the protein. The endogenous NHS-1A in fibroblasts is mainly a cytoplasmic protein. Similar to its localization, GFP–NHS(375–1652), deficient in the N-terminal 196 residues in NHS-1A, localized to the cytoplasm in all the cells tested in this study. However, definitive nuclear localization of the N-terminal approximately 200 amino acid region of Nhs-1A in Nhs1A–GFP was reported in CHO cells (15). Whether this nuclear localization is cell-type-dependent or an experimental artefact warrants investigation of the full-length NHS-1A in various cell types.
In situ hybridization shows Nhs expression throughout murine lens development, from lens placode to mature lens. It is expressed in the anterior lens vesicle cells, primary fibre cells and differentiated lens epithelium but not in secondary or mature lens fibres. Correspondingly, the Nhs protein was also detected in the lens epithelium but not in lens fibres in postnatal rat lens (Fig. 10A). Contrary to our findings, Huang et al. (15) detected Nhs protein in fibre cells of mouse lens. These contrasting results may either be due to difference in the antibodies, their specificity and/or organisms used in the two studies. Huang et al. generated the antibody against a 272 amino acid C-terminal region of the mouse protein that has homology with the paralogous NHS-like1 protein. The antibody in the present study was generated against the C-terminus peptide unique to NHS and shared by the human, mouse, rat and dog NHS proteins. The in situ expression in mouse lens and immunolabelling in rat lens presented here concordantly demonstrate NHS expression in mammalian lens epithelium and not in fibre cells of developed lens. Furthermore, both mRNA and protein analysis showed that NHS-A isoform is expressed in human and mouse lens, and immunolabelling localized the protein to apical cell membrane in human and rat lens epithelium. This localization in vivo is consistent with the localization of NHS-A in epithelium ex vivo. Thus transcript and protein analysis in human, mouse and rat collectively demonstrate the expression of NHS in the lens epithelium and not in lens fibres.
The in vivo and ex vivo data taken together suggest a role for the NHS-A isoform at cell–cell contacts. On the basis of the bioinformatic analysis, Katoh and Katoh (24) proposed NHS to be the mammalian orthologue of Drosophila guanylate kinase holder (GUKH), a scaffolding protein at neuromuscular junctions that mediates the interaction between PDZ (PSD-95/Dlg/ZO-1) proteins, discs large (DLG) and scribble (SCRIB) (25). NHS exhibits 10% amino acid identity with GUKH. The mammalian Dlg and Scrib proteins, implicated in regulating epithelial cell polarity, are present at adherens junctions in epithelial cells and co-localize with the adherens junction protein E-cadherin (26–28). The adherens junctions are basal to TJs in cell membrane of polarized epithelium but NHS-A is distributed at apical membrane and co-localizes with ZO-1 (Fig. 11) not E-cadherin (Fig. 12) in epithelial cells. Therefore, the possibility of its interaction with Dlg and Scrib and functional similarity to Drosophila GUKH seem unlikely.
PredictProtein analysis suggested that the majority of NHS protein has no regular secondary structure (NORS) (29,30). Consistent with this prediction, both NHS-A and NHS-1A proteins migrate slower than expected in SDS–PAGE. The hydrophilic and flexible NORS regions in proteins are believed to acquire functional conformational upon binding to their substrate or ligand (31). Up to 30% of eukaryotic proteins have NORS regions and due to their conformational adaptability may bind to many different targets. NORS regions are found more often in regulatory proteins and transcription factors. The presence of NORS regions in NHS and localization of its isoforms to multiple cellular sites fit well with the possibility of interaction of NHS with diverse targets and involvement in regulation of cellular processes. Regions of homology to actin-binding proteins dynamin and synaptojanin and to WH1 domain in WAVE and WASP, required for interaction with actin remodelling proteins, found in Nhs (15) also point to its potential interaction with various proteins. Localization of the NHS-A isoform at cell–cell contacts in mammalian epithelium and homology of mouse Nhs to proteins associated with the actin cytoskeleton open the possibility of involvement of NHS-A in the regulation of cellular architecture.
In conclusion, we highlight the emerging complexity of the regulation of the NHS gene expression. The presence of several isoforms and their localization to different parts of the cell indicates their ability to perform diverse functions possibly through interaction with a wide range of protein molecules. Potential involvement of NHS in diverse cellular processes may be the cause of pleiotropic features in the Nance–Horan syndrome. The NHS gene is expressed throughout lens development and disruption of its expression or translation leads to cataract formation in humans and mice; hence, it is a pivotal gene in ocular lens development. The NHS-A isoform expressed in the lens epithelium localizes with intercellular contacts and disruption of the latter is believed to underlie cataractogenesis in the Nance–Horan syndrome; however, this hypothesis is yet to be experimentally tested. Further understanding of the function performed by the other NHS isoforms and their role in mammalian development may explain the pleiotropic features of the syndrome.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mammalian cell lines and cell culture
The cell lines used in this study were kindly provided by Drs Venkat Reddy, Kellogg Eye Institute, MI, USA (SRA 01/04 human lens epithelium), Paul Russell, National Eye Institute, MD, USA (
TN4 mouse lens epithelium) and Stephen A. Wood, Child Health Research Institute, North Adelaide, South Australia, Australia (MDCK). The HEK 293A cells were from Qbiogene and SH-SY5Y human neuroblastoma cells from the American Type Culture Collection, VA, USA. All cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin in a humidified atmosphere at 37°C and 5% CO2. Cultures of SH-SY5Y cells were grown in a 1:1 mixture of DMEM and Ham's F12 medium supplemented with 10% fetal bovine serum.
Cloning of full-length coding cDNA
The first 1368 coding nucleotides of NHS cDNA were amplified from SRA 01/04 human lens epithelium cDNA as a single PCR product using GC-RICH PCR system (Roche Diagnostics) and gene-specific 5'-TACCGGAATTCTCCTTTCGCCAAGCGGATCGTGGAG-3' forward and 5'-CAGAATATCCTCGGTTTGGCACTCAGAG-3' reverse primers. The overlapping 3834 coding nucleotides including the termination codon were amplified from human hippocampal cDNA as two overlapping PCR products. The first of these two products was amplified with 5'-CCGCTCGAGACAGAGAGGCTAGTATACGC-3' forward and 5'-GATGCCAAGCCAGCCAGC-3' reverse primers, and the second with 5'-GAGCCACCACCCCATCTC-3' forward and 5'-CGGGTCGACCTATGTTGAACTCTGGGAGG-3' reverse primers using Expand Long Range Enzyme Mix (Roche Diagnostics). The three resulting PCR products were ligated in tandem using the restriction sites in the overlapping regions to create the complete coding cDNA. The resulting clone was verified by sequencing.
Antibody production
Rabbits were immunized with the C-terminus peptide CSDGSPHDDRSSQSST in complete Freund's adjuvant for primary injection and in incomplete Freund's adjuvant for booster injections. Serum was collected from immune blood and antibody affinity purified on peptide-conjugated Thiopropyl Sepharose 6B column (Mimotopes) as per manufacturer's protocol.
Generation of fusion constructs
EcoRI/SalI-blunt fragment including the complete NHS-A coding cDNA without initiation codon was cloned into EcoRI/SmaI-digested pEGFP-C1 vector (Clontech) to create the GFP–NHS-A construct. EcoRI/EcoRV cDNA fragment encoding 2–442 residues of NHS-A was cloned into pEGFP-C1 at EcoRI/SmaI to generate the GFP–NHSA442 constructs. GFP–NHS(375–1652) construct was generated by cloning the cDNA encoding the C-terminal 1278 amino acids of NHS at XhoI/SalI in pEGFP-C1.
Expression analysis
Postmortem human retina and lens were obtained through the Eye Bank of South Australia following ethical guidelines of the Clinical Research and Drug Trial Committee, Flinders Medical Centre, Australia. RNA from cultured mammalian cells and human tissues was extracted with the RNAeasy mini kit (Qiagen) as per manufacturer's protocol. Human fetal and adult brain RNA was from Clontech. First-strand cDNA synthesis was performed with Superscript III (Invitrogen) using random hexamers. RT–PCR for NHS-A was carried out with 5'-TGCAGCCTCTTCCAGGAGCTCGAGA-3' forward and 5'-ATTGGGTTTTTCGGCCTCTGCCCTA-3' reverse primers and HotStar Taq (Qiagen) at 95°C for 15 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s for 40 cycles. NHS was similarly amplified with 5'-GAGACCCAAGGAAATGTGGA-3' forward and 5'-ATGTCCCCGGAATCTTTTCT-3' reverse (human) or 5'-GAGACCCAAGGAAGTATGGA-3' forward and 5'-ATATCCCCAGAATCTTTTCT-3' reverse (mouse) primers at 60 and 52°C annealing, respectively, for 35–40 cycles. Amplified products were analysed by agarose gel electrophoresis.
In situ hybridization was performed on paraffin sections of the developing mouse lens as described elsewhere (32). Sections were hybridized with digoxigenin-labelled antisense and sense riboprobes generated from 511 bp region of Nhs in exon 6 as reported previously (10).
Western blotting
Mouse lens tissue was obtained following ethical guidelines of the Animal Welfare Committee, Flinders University, Australia. Protein extracts from frozen cell pellets, transiently transfected HEK 293A cells and lens tissue were prepared in lysis buffer [PBS, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, Protease Inhibitor Cocktail (Roche Diagnostics), PMSF and 0.1% ß-mercaptoethanol]. Forty micrograms of total soluble protein was size fractionated by SDS–PAGE and transferred onto Hybond-C extra (Amersham Biosciences). Western blots were probed with 1:200 dilution of affinity purified anti-NHS antibody and 1:20 000 dilution of anti-rabbit IgG-HRP secondary antibody (Rockland Immunochemicals) or 1:500 dilution of anti-GFP mouse monoclonal antibody (Roche Diagnostics) and 1:1000 dilution of sheep anti-mouse Ig-HRP secondary antibody (Chemicon). Antibody binding was detected with ECL or Advance ECL kit (GE Biosciences).
Immunofluorescent labelling
About 3x105 MDCK cells were seeded per 22 mm2 cover slip and cultured for 4–5 days. NIH 3T3 and SH-SY5Y cells seeded at a density of 1–2x105 cells per cover slip were cultured for 48 h. For immunolabelling, cells were fixed in 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, blocked with 5% donkey serum and hybridized with affinity purified anti-NHS primary antibody and fluorescein (FITC)-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories). For co-localization with ZO-1, MDCK cells were hybridized with anti-NHS and mouse anti-ZO-1 (Zymed Laboratories) primary antibodies followed by FITC-conjugated donkey anti-rabbit IgG and Cy3-conjugated donkey anti-mouse IgG (Rockland Immunochemicals) secondary antibodies. For co-localization with E-cadherin, MDCK cells were hybridized with anti-NHS and mouse anti-E-cadherin (BD Transduction Laboratories) primary antibodies followed by Cy5-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories) secondary antibodies. After labelling, cells were mounted on slides in buffered glycerol. Confocal microscopy was performed on an Olympus AX70 microscope attached to a Bio-Rad 1024 MRC scanning confocal system equipped with an Argon Ion and a Helium Neon laser using LaserSharp 2000 software. FITC was excited with 488 nm laser line and detected at 522 nm, Cy3 excited with 568 nm laser line and detected at 615 nm and Cy5 excited with 647 nm laser line and detected at 680 nm.
Sections of methanol-fixed lenses from P1 and 11 Wistar rats were immunolabelled with anti-NHS antibody and detected with FITC-conjugated anti-rabbit IgG secondary antibody (Molecular Probes). Nuclei were stained with propidium iodide (PI). Confocal microscopy was performed with the LSM PASCAL (Zeiss) equipped with an argon ion and a helium–neon laser and using LSM imaging software. FITC was excited with 488 nm laser line and detected at 594 nm, and PI was excited with 543 nm laser line and detected at 617 nm.
Transfection of mammalian cells with GFP-fusion constructs
About 3x105 cells were seeded onto glass cover slips in six-well plates. HEK 293A and SRA 01/04 cells were transfected on the following day with GFP-fusion or control plasmid using FuGENE6 reagent (Roche Diagnostics) according to manufacturer's protocol. MDCK cells were transfected on the third day using Lipofectamine 2000 (Invitrogen) according to manufacturer's protocol. Approximately 48 h post-transfection, the cells were fixed in 4% paraformaldehyde and mounted on slides in buffered glycerol. Confocal microscopy was performed as described above. GFP was excited with 488 nm laser line and detected at 522 nm.
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ACKNOWLEDGEMENTS |
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Thanks are due to Ms Margaret Philpott, Co-ordinator, Eye Bank of South Australia, for obtaining consent from donor families and retrieving donor eye tissue for this work. We thank Professor Barry Powell, Child Health Research Institute, North Adelaide, SA, Australia, for sharing the ZO-1 antibody. This work was funded by the National Health and Medical Research Council (NHMRC) of Australia. J.E.C. is a recipient of the NHMRC practitioner fellowship. A part of this work was presented at the ComBio2005, Adelaide, Australia, and the AOVS Meeting 2005, Melbourne, Australia.
Conflict of Interest statement. There is no conflict of interest to be declared.
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