Usher syndrome and Leber congenital amaurosis are molecularly linked via a novel isoform of the centrosomal ninein-like protein - PubMed (original) (raw)

. 2009 Jan 1;18(1):51-64.

doi: 10.1093/hmg/ddn312. Epub 2008 Sep 30.

Ferry F J Kersten, Aileen Kartono, Dorus A Mans, Kim Brandwijk, Stef J F Letteboer, Theo A Peters, Tina Märker, Xiumin Yan, Cor W R J Cremers, Frans P M Cremers, Uwe Wolfrum, Ronald Roepman, Hannie Kremer

Affiliations

Usher syndrome and Leber congenital amaurosis are molecularly linked via a novel isoform of the centrosomal ninein-like protein

Erwin van Wijk et al. Hum Mol Genet. 2009.

Abstract

Usher syndrome (USH) and Leber congenital amaurosis (LCA) are autosomal recessive disorders resulting in syndromic and non-syndromic forms of blindness. In order to gain insight into the pathogenic mechanisms underlying retinal degeneration, we searched for interacting proteins of USH2A isoform B (USH2A(isoB)) and the LCA5-encoded protein lebercilin. We identified a novel isoform of the centrosomal ninein-like protein, hereby named Nlp isoform B (Nlp(isoB)), as a common interactor. Although we identified the capacity of this protein to bind calcium with one of its three EF-hand domains, the interacton with USH2A(isoB) did not depend on this. Upon expression in ARPE-19 cells, recombinant Nlp(isoB), lebercilin and USH2A(isoB) were all found to co-localize at the centrosomes. Staining of retinal sections with specific antibodies against all three proteins revealed their co-localization at the basal bodies of the photoreceptor-connecting cilia. Based on this subcellular localization and the nature of their previously identified binding partners, we hypothesize that the pathogenic mechanisms for LCA and USH show significant overlap and involve defects in ciliogenesis, cilia maintenance and intraflagellar and/or microtubule-based transport. The direct association of Nlp(isoB) with USH2A(isoB) and lebercilin indicates that Nlp can be considered as a novel candidate gene for USH, LCA and allied retinal ciliopathies.

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Figures

Figure 1.

Figure 1.

Schematic representation of the protein structure of NlpisoA and NlpisoB. (A) The predicted EF hands 1, 2 and 3 are formed by amino acids 11–39, 200–228 and 237–265, respectively. Coiled-coil domains (CC) 1, 2, 3 and 4 are formed by amino acids 384–425, 470–579, 621–699 and 1046–1375, respectively, and the predicted IF domain is formed by amino acids 656–925. (B) Semi-quantitative RT–PCR for transcripts encoding NlpisoA and NlpisoB in human fetal and adult tissues. Shown are the samples that were taken after 35 cycles. The results are representative for the samples taken after 25 and 30 cycles. As a control, RT–PCR analysis of the household gene GAPDH was performed. The transcripts for NlpisoA and NlpisoB show a similar distribution, with the strongest expression in fetal cochlea and adult brain, testis and kidney.

Figure 2.

Figure 2.

Protein–protein interaction studies. (A) The schematic protein structure of NlpisoB and protein fragments encoded by deletion constructs with the corresponding amino acids are depicted. Yeast two-hybrid analysis showed a specific interaction between the fragment of the intracellular region of USH2AisoB encompassing amino acids 5124–5196 and the predicted IF domain (amino acids 656–925) of NlpisoB. (B) Schematic representation of the protein structure of lebercilin and protein fragments encoded by deletion constructs with the corresponding amino acids. Yeast two-hybrid analysis revealed a specific interaction between the lebercilin domain containing the two N-terminal coiled-coil domains (amino acids 96–305) and the predicted IF domain of NlpisoB (amino acids 656–925). A liquid β-galactosidase assay revealed a specific interaction between NlpisoB and the USH2A_tail (C) and NlpisoB and lebercilin (D). No interaction was observed between USH2A and NlpisoA (C) and lebercilin and NlpisoA (D). aa, amino acids; NA, not assayed.

Figure 3.

Figure 3.

Co-immunoprecipitation of NlpisoB with the intracellular region of USH2AisoB (USH2A_tail), but not with STRAD. (A) The immunoblot (IB) in the left upper panel shows that HA-NlpisoB (lane 1) co-immunoprecipitated with flag-USH2A_tail, but not with the unrelated protein flag-STRAD (lane 2). Protein input is shown in the left lower panel; the anti-flag immunoprecipitates are shown in the right panel. (B) GST pull-down assays showing that flag-tagged lebercilin was efficiently pulled down by GST-fused NlpisoB, but not by GST alone. The left lane shows 2% input of the protein lysate. (C) Co-immunoprecipitation of lebercilin with NlpisoB, but not with LRRK2. The immunoblot (IB) in the right panel shows that flag-NlpisoB (right lane) co-immunoprecipitated with HA-lebercilin, whereas the unrelated protein flag-LRRK2 (left lane) did not. Protein input is shown in the left panel; the anti-HA immunoprecipitates are shown in the middle panel. (D) Co-immunoprecipitation of endogenous Nlp with lebercilin from bovine retinal extracts, but not with rabbit IgGs. The top panel shows that Nlp was co-immunoprecipitated with lebercilin, indicated by an arrow, whereas it was not with rabbit IgGs. The bottom panel shows that lebercilin was immunoprecipitated with lebercilin-specific antibodies, as indicated by an arrow, but not with rabbit IgGs.

Figure 4.

Figure 4.

Analysis of Ca2+-binding properties of Nlp. (A) Multiple protein alignment of the predicted EF-hand domains in Nlp and the consensus sequence for EF-hand domains (SMART database). The invariable D or E at position 12 is boxed and indicated by an arrow and is only present in EF hand 3. (B) A calcium-binding assay showing the specific calcium-binding capacities of Nlp EF hand 3. The 28K subunit of calmodulin was used as a positive control. (C) GST pull-down analysis showing that HA-tagged NlpisoB was efficiently pulled down by GST-fused USH2A_tail in the presence and absence of calcium, but not by GST-NBC3_tail and GST alone. Lanes 1 and 5 show 5% of the input protein lysate.

Figure 5.

Figure 5.

Co-localization of Nlp and USH2A in the retina. (A–A″) Co-immunostaining of USH2A and Nlp in radial cryosections of adult (P20) rat retina by using anti-Nlp antibodies (green signal; A) and anti-USH2A antibodies (red signal; A′) showing co-localization (yellow signal; A″) in the inner segment (IS) and in the region of the connecting cilium (CC). (B) High magnification fluorescence microscopy analysis of double immunofluorescence with anti-Nlp (green) and anti-_pan_-centrin antibodies (red; marker for the ciliary apparatus: connecting cilium, centriole and basal body) in cryosections through the ciliary part of rat photoreceptor cells. Merged images indicate partial co-localization of Nlp with centrins in the centriole and basal body (yellow signal). Pre-embedding immunolabellings of the ciliary region of mouse photoreceptors by antibodies against Nlp (C) and USH2AisoB (D) show a clear staining of the (apical part of) inner segment (IS), the centriole (indicated by an asterisk) and the basal body (BB).

Figure 6.

Figure 6.

Centrosomal localization of NlpisoB, USH2A_tail and lebercilin in ARPE-19 cells. When expressed alone, mRFP–NlpisoB (red signal) was localized to the mother centriole of the centrosome (A), eCFP-USH2A_tail (green signal) was localized to the nucleus and the centrosome (indicated by an arrow and in the inlay) (B), whereas eYFP-lebercilin (green signal) was localized at the centrosome and the microtubule network of the cell (C). After co-expression of NlpisoB and USH2A, both proteins were localized at the mother and daughter centriole of the centrosome, confirming the interaction between both proteins (D–D″). Co-expression of NlpisoB and lebercilin showed co-localization of both proteins at the centrosome and non-centrosomal MTOCs (yellow signal; E″). Lebercilin in addition partly localized at the microtubule network (E–E″). Nuclei were stained with DAPI (blue signal).

Figure 7.

Figure 7.

Localization of endogenously expressed Nlp and lebercilin in ARPE-19 cells by immunocytochemistry using the anti-Nlp and anti-lebercilin antibodies (green signals) and anti-acetylated tubulin antibodies as an axoneme and microtubule marker (red signal). Nlp (A) and lebercilin (B) were present at the basal body of primary cilia and both proteins were found at the midbody region of ARPE-19 cells during telophase (C and D). Nuclei were stained with DAPI (blue signal).

Figure 8.

Figure 8.

Mutations in lebercilin affect the interaction with NlpisoB. A quantitative liquid β-galactosidase assay shows an enhanced interaction of NlpisoB and lebercilinQ279X when compared with wild-type lebercilin and a reduced interaction of NlpisoB and lebercilinP493TfsX1 (A). When expressed alone in ARPE-19 cells, mRFP–lebercilin wild-type (red signal) was localized to the basal body, the primary cilium and the microtubule network of the cell (B), mRFP-lebercilinQ279X was localized to the basal body and primary cilium (C) and mRFP-lebercilinP493TfsX1 was localized to the microtubule network of the cell (D). Co-expression of NlpisoB and lebercilin showed co-localization of both proteins at the basal body (yellow signal; E″, inset). In addition, lebercilin localized to the primary cilium and the microtubule network in the cell periphery (red signal; E′–E″, inset). Upon co-expression of NlpisoB and lebercilinQ279X, both proteins co-localize at the basal body (yellow signal; F″, inset). The mutant lebercilin was not found in the primary cilium (F′–F″, inset). After co-expression of NlpisoB and lebercilinP493TfsX1, no co-localization of these proteins was observed (G–G″, inset). Nuclei were stained with DAPI (blue signal).

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