The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis

Abstract

In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development. In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development. Cilia are evolutionarily conserved microtubule-based organelles that extend from the basal bodies and are formed on the cell surface (1Gerdes J.M. Davis E.E. Katsanis N. The vertebrate primary cilium in development, homeostasis, and disease.Cell. 2009; 137: 32-45Google Scholar, 2Malicki J.J. Johnson C.A. The cilium: Cellular antenna and central processing unit.Trends Cell Biol. 2017; 27: 126-140Google Scholar). They are generally classified into two categories: motile cilia and primary cilia (3Ishikawa H. Marshall W.F. Ciliogenesis: Building the cell's antenna.Nat. Rev. Mol. Cell Biol. 2011; 12: 222-234Google Scholar). Motile cilia are formed on specific epithelial cell types and cooperatively beat in wave-like patterns to generate fluid flow. Sperm flagella are an example of motile cilia which mainly function in cell locomotion. On the other hand, primary cilia are present in almost every cell type in vertebrates and act as cellular antennae to sense physical and biochemical extracellular signals. In humans, ciliary dysfunction causes diseases known as ciliopathies, which are characterized by a broad spectrum of pathologies, including polydactyly, craniofacial abnormalities, brain malformation, situs inversus, obesity, diabetes, retinal and renal degeneration, hearing loss, and infertility (4Nigg E.A. Raff J.W. Centrioles, centrosomes, and cilia in health and disease.Cell. 2009; 139: 663-678Google Scholar, 5Hildebrandt F. Benzing T. Katsanis N. Ciliopathies.N. Engl. J. Med. 2011; 364: 1533-1543Google Scholar). Many ciliary proteins are known to play important roles in human physiology, signaling, and development (6Reiter J.F. Leroux M.R. Genes and molecular pathways underpinning ciliopathies.Nat. Rev. Mol. Cell Biol. 2017; 18: 533-547Google Scholar). It was recently reported that there are at least 38 established ciliopathies with mutations in at least 247 genes (7Lovera M. Luders J. The ciliary impact of nonciliary gene mutations.Trends Cell Biol. 2021; 31: 876-887Google Scholar). For example, Bardet–Biedl syndrome (BBS), which is an autosomal recessive ciliopathy that results in a plethora of developmental and multiorgan defects, is known to be caused by mutations in 22 identified pathogenic genes (8McConnachie D.J. Stow J.L. Mallett A.J. Ciliopathies and the kidney: A review.Am. J. Kidney Dis. 2021; 77: 410-419Google Scholar). In addition, mutations in more than 25 different pathogenic genes have been shown to be associated with nephronophthisis (NPHP), an autosomal recessive renal ciliopathy with additional features such as retinal defects, liver fibrosis, skeletal abnormalities, and brain developmental disorders (9Luo F. Tao Y.H. Nephronophthisis: A review of genotype-phenotype correlation.Nephrology (Carlton). 2018; 23: 904-911Google Scholar). Although many gene mutations causing ciliopathies have been found in humans, the molecular and pathological mechanisms underlying ciliopathies are still not well understood. Serologically defined colon cancer antigen 8 (SDCCAG8) was identified as a centrosome-associated tumor antigen protein and is also known as centrosomal colon cancer autoantigen protein, NPHP10, and BBS16 (10Kenedy A.A. Cohen K.J. Loveys D.A. Kato G.J. Dang C.V. Identification and characterization of the novel centrosome-associated protein CCCAP.Gene. 2003; 303: 35-46Google Scholar). Mutations in SDCCAG8 are associated with an NPHP-related ciliopathy, characterized by retinal and renal degeneration, cognitive defects, obesity, hypogonadism, hearing loss, recurrent respiratory infections, and infrequently clinodactyly (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar, J. M. F. M. in syndrome and are associated with renal and 2011; Scholar, of SDCCAG8 in syndrome patients with renal and Scholar, T. N. T. M. M. H. renal ciliopathies in that were identified by 2017; Scholar, N. nephronophthisis in a with a SDCCAG8 J. Med. Scholar, M. M. H. A novel in the SDCCAG8 gene in an with 2021; Scholar). have reported that Sdccag8 is for cilia formation and F. SDCCAG8 and in the Scholar, M. M. J. Hurd J.M. F. SDCCAG8 interacts with proteins and and is for Scholar, M. gene regulation as a by which ciliopathy gene SDCCAG8 to and cognitive Mol. Scholar). Sdccag8 mice with multiple organ defects, and Sdccag8 mice exhibited retinal and renal F. SDCCAG8 and in the Scholar, N. Hurd J.M. M. ciliopathy gene Sdccag8 Scholar). It also been reported that Sdccag8 is associated with the regulation of F. SDCCAG8 and in the Scholar). mutations in the human SDCCAG8 gene that are predicted to truncate the Sdccag8 carboxyl (C) terminus are associated with multiple organ defects, including retinal and cystic (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar, J. M. F. M. in syndrome and are associated with renal and 2011; Scholar, of SDCCAG8 in syndrome patients with renal and however, the underlying pathological mechanisms are poorly understood. We and have previously reported that cell also known as and play roles in the regulation of cilia length and ciliary in mammals T. N. T. M. T. regulation of ciliary length by ciliary is for retinal Scholar, T. T. is essential for cell and the regulation of ciliary J. Scholar, H. J. H. J. cell a protein associated with is a of cilia length and Scholar, K.J. of cilium length and by the and in renal epithelial Scholar, T. H. T. ciliary is essential for cell formation in and hearing 2017; Scholar, T. T. as of ciliary protein 2021; Scholar). and are that to the protein and in of and Scholar, T. cell to the region and a found in Cell Scholar, H. N. M. Kato T. T. A is for Biol. Scholar). is in is in the and T. T. of the ciliopathy gene in cell of 2018; Scholar). mutations in the human gene have been reported to to syndrome and recessive ciliopathies with multiple developmental abnormalities, including cleft and cystic J. J.F. D.A. C.A. A human syndrome a for cell in development of central and J. 2009; Scholar, D.A. H. C.A. G.J. novel causes ciliary and Scholar, M. M. T. M. of for in cell and causes Mol. Scholar). Mutations in the human gene also autosomal recessive J.M. and of the gene as a of 2011; Scholar, C.A. and mutations in a gene a of ciliary as a of J. 2011; Scholar). In the study, we found that and with the C-terminal region of Sdccag8 (Sdccag8-C) and the of Sdccag8-C in and in the molecular mechanisms of cilia development and ciliopathies, we for proteins that with protein using and identified proteins, including Sdccag8 protein previously been associated with ciliopathies with multiple organ as well as we on Sdccag8 in the present we the interaction and Sdccag8 by using with an the interaction and we an using a cell with an We observed the in with the by the interaction and The also an interaction and Sdccag8 SDCCAG8 is a ciliary protein that of predicted and be into and (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar). which region of Sdccag8 interacts with and Mak, we mouse Sdccag8 into and and interaction with and by that and with the Sdccag8 and not and results that and with the Sdccag8 protein is known to to centrosomes and basal bodies (10Kenedy A.A. Cohen K.J. Loveys D.A. Kato G.J. Dang C.V. Identification and characterization of the novel centrosome-associated protein CCCAP.Gene. 2003; 303: 35-46Google Scholar, E.A. Hurd M. C.A. J. of SDCCAG8 as the of a we the of Sdccag8-C in to We the protein the to region of mouse Sdccag8 Sdccag8-C into and localization to centrosomes by with and for centrosomes and basal We observed that and Sdccag8-C to centrosomes in the of Sdccag8-C at centrosomes was than that of In of and were not at results that Sdccag8-C is for localization to the basal the localization of Sdccag8-C at basal we the a mouse the of the in vertebrate Sdccag8 proteins We into and localization to we not and at centrosomes, Sdccag8-C at centrosomes in results that the Sdccag8-C a that to basal the roles of Sdccag8-C in cilia we and using cells. We to Sdccag8 and that and the Sdccag8 We also that and the Sdccag8 in We these into and the cilia by using ciliary and We observed that and the of and cilia length we a human SDCCAG8 and the to region of human SDCCAG8 We the with into and the cilia by using and We found that the of the of cilia formation In the of to the of cilia formation. results that is for cilia formation. to the in roles of we generated Sdccag8 knock-in mice using the A stop codon and an were the Sdccag8-C in Sdccag8ΔC/ΔC mice The was to the of the stop codon We and that the of Sdccag8 were and Sdccag8ΔC/ΔC mice that Sdccag8 is not by in Sdccag8ΔC/ΔC mice. the of Sdccag8ΔC/ΔC mice, we the of the from at the from to and at from to The of Sdccag8ΔC/ΔC mice was than that of mice from to Sdccag8ΔC/ΔC mice were present at the at that Sdccag8ΔC/ΔC mice not In addition, we found that Sdccag8ΔC/ΔC mouse a cleft We also found that several Sdccag8ΔC/ΔC mice exhibited that Sdccag8-C is for proper skeletal development. Sdccag8ΔC/ΔC mice exhibited ciliopathy-like such as cleft and polydactyly, we cilia formation and function in Sdccag8ΔC/ΔC mice. the of Sdccag8-C in cilia formation and we cilia in mouse from Sdccag8ΔC/ΔC We Sdccag8 in Sdccag8ΔC/ΔC with an We observed that Sdccag8 at centrosomes in Sdccag8ΔC/ΔC were than those in and a localization of Sdccag8 at centrosomes in Sdccag8ΔC/ΔC We of using the and found that the of and cilia length in Sdccag8ΔC/ΔC with that in the of Sdccag8-C the protein in we of in the cilia of Sdccag8ΔC/ΔC We observed in the ciliary localization of and Sdccag8ΔC/ΔC Cilia are known to play important roles in Cilia and in the Scholar). the of Sdccag8-C signaling, we observed ciliary localization of a in Sdccag8ΔC/ΔC with of a J. T. of Scholar). of the with ciliary localization of at the primary Scholar). We observed in the localization to cilia and Sdccag8ΔC/ΔC and We the of a gene of the in Sdccag8ΔC/ΔC by The that in Sdccag8ΔC/ΔC with that in in the of the results that Sdccag8-C an important in cilia formation and the Sdccag8 in we using mouse at that Sdccag8 is in including the kidney, and Sdccag8 in the we in using retinal at We observed that Sdccag8 were in the the roles of Sdccag8-C in the we using retinal from and Sdccag8ΔC/ΔC mice. that there was the and Sdccag8ΔC/ΔC at however, the in the Sdccag8ΔC/ΔC A and In we observed in the of the other the and Sdccag8ΔC/ΔC results that Sdccag8ΔC/ΔC mice in the Sdccag8ΔC/ΔC we of retinal using for for and for and and of and in the were observed in the Sdccag8ΔC/ΔC at and and We also observed the of in the of the with the localization of we Sdccag8 and in the of the Sdccag8ΔC/ΔC and found that Sdccag8 at basal bodies in Sdccag8ΔC/ΔC were than those in and the localization of Sdccag8 at basal bodies in Sdccag8ΔC/ΔC we and observed that the of cilia in the Sdccag8ΔC/ΔC with that in the and we found that the of cilia in the Sdccag8ΔC/ΔC with that in the and On the other hand, we in cilia of the Sdccag8ΔC/ΔC and observed in the ciliary localization of and Sdccag8ΔC/ΔC and these results that Sdccag8-C is essential for cilia formation. the of Sdccag8-C we the of Sdccag8ΔC/ΔC mice and We observed that the of and of Sdccag8ΔC/ΔC mice and with those of mice at results that and function is by the Sdccag8-C the of Sdccag8-C in the kidney, we observed the of the Sdccag8ΔC/ΔC were observed in Sdccag8ΔC/ΔC mice at we and using renal from and Sdccag8ΔC/ΔC mice at and The formation in the Sdccag8ΔC/ΔC from to renal fibrosis, which are observed in patients with F. 18: in the Sdccag8ΔC/ΔC kidney, we using renal from and Sdccag8ΔC/ΔC mice at in the Sdccag8ΔC/ΔC results that Sdccag8ΔC/ΔC mice phenotypes, and the of Sdccag8-C to fibrosis, and renal It previously been reported that patients with mutations in SDCCAG8 gene and J. M. F. M. in syndrome and are associated with renal and 2011; however, the of Sdccag8 in the We the from Sdccag8ΔC/ΔC mice and observed that the of Sdccag8ΔC/ΔC mice were than those of mice and into and Cell in the cell and Rev. Scholar). the roles of Sdccag8 in the we using from and Sdccag8ΔC/ΔC mice. a of with in the of the Sdccag8ΔC/ΔC at that of is in the Sdccag8ΔC/ΔC In we observed and in the Sdccag8ΔC/ΔC that the and in the Sdccag8ΔC/ΔC are In addition, and were also observed in the Sdccag8ΔC/ΔC our results that Sdccag8-C is for Sdccag8 may play a in flagella formation. In the study, we and found that and with the roles of Sdccag8-C in we generated Sdccag8ΔC/ΔC mice, in which Sdccag8-C was by the CRISPR-mediated stop codon knock-in strategy. We observed ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal and renal degeneration, and in Sdccag8ΔC/ΔC mice, there may be in other we also found ciliary abnormalities in Sdccag8ΔC/ΔC mice, that Sdccag8-C is for cilia formation and that Sdccag8 mice and that Sdccag8 mice are present at at F. SDCCAG8 and in the Scholar, N. Hurd J.M. M. ciliopathy gene Sdccag8 Scholar). Sdccag8 mice and Sdccag8 mice, Sdccag8ΔC/ΔC mice exhibited Together, our and previous that Sdccag8ΔC/ΔC mice be as a and mouse to the pathological mechanisms underlying the ciliopathies associated with mutations in SDCCAG8 In the Sdccag8ΔC/ΔC we observed a of with in that Sdccag8-C is for previous have shown that Sdccag8 is in and in the T. M. of the centrosomal colon cancer autoantigen gene in the Sdccag8 may play a in flagella formation in a cell we the that dysfunction of by the of Sdccag8-C to the in the Sdccag8ΔC/ΔC Furthermore, we have to the dysfunction of the in Sdccag8ΔC/ΔC mice. into the pathological mechanisms underlying and in patients with mutations in SDCCAG8 gene and the roles of Sdccag8 in the the molecular and pathological mechanisms of Sdccag8 dysfunction in the is Sdccag8-C involved in cilia formation and We found that Sdccag8-C interacts with and proteins by We previously found that mainly to the ciliary however, several have reported that also to the ciliary T. T. is essential for cell and the regulation of ciliary J. Scholar, H. J. H. J. cell a protein associated with is a of cilia length and Scholar, K.J. of cilium length and by the and in renal epithelial Scholar). Furthermore, we previously found that to the cilia and ciliary as well as basal bodies in retinal T. N. T. M. T. regulation of ciliary length by ciliary is for retinal Scholar). that Sdccag8 to centrosomes and basal bodies (10Kenedy A.A. Cohen K.J. Loveys D.A. Kato G.J. Dang C.V. Identification and characterization of the novel centrosome-associated protein CCCAP.Gene. 2003; 303: 35-46Google Scholar, E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Sdccag8 is to with and at basal We found a in cell cilia and in Sdccag8ΔC/ΔC in Sdccag8ΔC/ΔC were observed in T. T. is essential for cell and the regulation of ciliary J. Scholar). In addition, retinal in Sdccag8ΔC/ΔC mice the of and which were also observed in the Mak-deficient T. N. T. M. T. regulation of ciliary length by ciliary is for retinal Scholar). In Ick- and Mak-deficient mice, Sdccag8ΔC/ΔC mice and in the ciliary localization of and and have been to as of at the ciliary and cilia length in mammals T. N. T. M. T. regulation of ciliary length by ciliary is for retinal Scholar, T. T. is essential for cell and the regulation of ciliary J. Scholar, H. J. H. J. cell a protein associated with is a of cilia length and Scholar, K.J. of cilium length and by the and in renal epithelial Scholar, T. H. T. ciliary is essential for cell formation in and hearing 2017; Scholar, T. T. as of ciliary protein 2021; Scholar). an of and Mak, was to cilia length and of than T. A and a cilia and length regulation of in Biol. Scholar). and may cilia length of interaction with Sdccag8 at the ciliary In humans, mutations in and genes have been reported to to retinal degeneration, cystic abnormalities, and respiratory J. J.F. D.A. C.A. A human syndrome a for cell in development of central and J. 2009; Scholar, D.A. H. C.A. G.J. novel causes ciliary and Scholar, M. M. T. M. of for in cell and causes Mol. Scholar, J.M. and of the gene as a of 2011; Scholar, C.A. and mutations in a gene a of ciliary as a of J. 2011; which are also associated with human SDCCAG8 mutations (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar, J. M. F. M. in syndrome and are associated with renal and 2011; Scholar, of SDCCAG8 in syndrome patients with renal and Scholar, T. N. T. M. M. H. renal ciliopathies in that were identified by 2017; Scholar, N. nephronophthisis in a with a SDCCAG8 J. Med. Scholar, M. M. H. A novel in the SDCCAG8 gene in an with 2021; Scholar). In addition, pathogenic in the gene have been to T. H. M. J.M. in Engl. J. Med. 2018; Scholar). A previous reported that a with a in the SDCCAG8 gene T. N. T. M. M. H. renal ciliopathies in that were identified by 2017; Scholar). our and previous that Sdccag8 interacts with and the identified other proteins that with the protein, we the that and and function with other proteins at basal We observed that and Sdccag8-C to centrosomes in of and not be at centrosomes, that Sdccag8-C as a module at basal We also observed localization of Sdccag8 to centrosomes basal bodies in Sdccag8ΔC/ΔC and cells. Sdccag8-C play roles in the localization of Sdccag8 to basal Sdccag8-C of that are associated with protein–protein (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar, J. A protein Cell Biol. Scholar). centrosomal/basal body proteins to the basal bodies J.M. to cilia 27: Scholar, J. is for ciliary to the Scholar, H. J.F. is for and ciliary in Scholar). It is that Sdccag8 to basal bodies the interaction of Sdccag8-C with centrosomal/basal body For example, previous reported that interacts with syndrome a centrosomal/basal body protein associated with and (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar, M. F. of the gene for type J. Scholar, M. M. T. is in syndrome and interacts with J. 2009; Scholar, D.A. M. N. N. M. in identified by causes a of Mol. Scholar). In addition, was previously reported that interacts with protein that to basal bodies and is for cilia formation M. M. J. Hurd J.M. F. SDCCAG8 interacts with proteins and and is for Scholar). that Sdccag8 also interacts with and Mak, Sdccag8 may play a as the of the body protein–protein interaction In human SDCCAG8 mutations that are predicted to truncate the Sdccag8 terminus were reported to be associated with multiple organ defects, including retinal and cystic (11Otto E.A. Hurd M. C.A. J. of SDCCAG8 as the of a Scholar, J. M. F. M. in syndrome and are associated with renal and 2011; Scholar, of SDCCAG8 in syndrome patients with renal and Scholar). results that Sdccag8-C is an Ick- and module that is for the localization of Sdccag8 to bodies and is essential for cilia formation and organ development and The shed light on the pathological mechanisms underlying the ciliopathies associated with mutations in SDCCAG8 on the Sdccag8 and the and advance our understanding of the molecular and pathological mechanisms underlying the to the for the of in and and these were by the on and the of for and were in with the were in a at 22 with a and were at Sdccag8ΔC/ΔC mice were generated using the The was to of the Sdccag8 gene using an codon and an was also with on of the The and in a were at for and to The was with at for and then with in The and were into the of mice. The mouse was then with the mice. and were previously T. N. T. M. T. regulation of ciliary length by ciliary is for retinal Scholar, T. T. is essential for cell and the regulation of ciliary J. Scholar). of mouse and Sdccag8 were by using mouse retinal as a and into the and Kato T. T. cell and in with in the mouse Biol. Scholar). The the mouse and Sdccag8-C were by using mouse retinal as a and into the The the mouse and were by using the as a and into the A of human SDCCAG8 was by using human retinal as a and into the The the human was by using the human SDCCAG8 as a and into the The was by using the as a and into the For Sdccag8 the and was into The were as T. T. an Scholar). For the of and the was into the T. H. T. M. M. T. T. T. function by Scholar). for are shown in was as previously T. T. T. N. T. a retinal protein, formation in and 2018; Scholar). In we a using the The of mouse was into the and into the We from a of mouse and the retinal were identified on the of to the and by on defined The were from using and into and Sdccag8ΔC/ΔC were from and cultured in with and at with in were to to and for were with for in and were cultured in and with and at with was with the for for cells. in the was by at the and were cultured for in were as previously T. T. J. Scholar). were with and of the were in a with and The cell were at for The were with an at The were with and and then with for at The were with for at and by For from cell the were in a with and and The cell and were at and then were for at with A The were with The were with for at and by was as previously T. M. H. T. T. H. H. T. is a cell essential for retinal 2017; Scholar). were in a with and with and and The were by and to using The were with and in were using We the primary T. 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