Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease
2014; Elsevier BV; Volume: 94; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2013.12.003
ISSN1537-6605
AutoresF. Buket Basmanav, Ana‐Maria Oprişoreanu, Sandra M. Pasternack, Holger Thiele, G. Fritz, Joerg Wenzel, Leopold Größer, Maria Wehner, Sabrina Wolf, Christina Fagerberg, Anette Bygum, Janine Altmüller, Arno Rütten, Laurent Parmentier, Laila El Shabrawi‐Caelen, Christian Hafner, Peter Nürnberg, Roland Kruse, Susanne Schoch, S. Hanneken, Regina C. Betz,
Tópico(s)RNA modifications and cancer
ResumoDowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4∗), c.652C>T (p.Arg218∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology. Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4∗), c.652C>T (p.Arg218∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology. Dowling-Degos disease (DDD [MIM 179850, MIM 615327]) is an autosomal-dominant form of a reticulate pigmentary disorder. This rare genodermatosis was first described by Dowling and Freudenthal in 19381Dowling G.B. Freudenthal W. Acanthosis Nigricans.Proc. R. Soc. Med. 1938; 31: 1147-1150PubMed Google Scholar and was termed “dermatose reticulée des plis” by Degos and Ossipowski (1954).2Degos R. Ossipowski B. Dermatose pigmentaire ŕeticulée des plis (discussion de l’acanthosis nigricans).Ann. Dermatol. Syphiligr. (Paris). 1954; 81: 147-151PubMed Google Scholar Affected individuals develop a postpubertal reticulate hyperpigmentation that is progressive and disfiguring, and small hyperkeratotic dark-brown papules that affect the flexures, large skin folds, trunk, face, and extremities. Pruritus and/or burning sensations might also feature in clinical presentations.3Gilchrist H. Jackson S. Morse L. Nicotri T. Nesbitt L.T. Galli-Galli disease: A case report with review of the literature.J. Am. Acad. Dermatol. 2008; 58: 299-302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The phenotype can be triggered in some individuals by UV light, mechanical stimulation, or sweating. Histology shows filiform epithelial downgrowth of epidermal rete ridges, with a concentration of melanin at the tips.4Betz R.C. Planko L. Eigelshoven S. Hanneken S. Pasternack S.M. Bussow H. Van Den Bogaert K. Wenzel J. Braun-Falco M. Rütten A. et al.Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease.Am. J. Hum. Genet. 2006; 78: 510-519Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar No effective therapy is yet available. In 2006, we identified loss-of-function mutations in KRT5 (MIM 148040) encoding keratin 5, in two large German families, additional familial cases, and several simplex cases.4Betz R.C. Planko L. Eigelshoven S. Hanneken S. Pasternack S.M. Bussow H. Van Den Bogaert K. Wenzel J. Braun-Falco M. Rütten A. et al.Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease.Am. J. Hum. Genet. 2006; 78: 510-519Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar Additional mutations in KRT5 responsible for DDD were also reported.5Guo L. Luo X. Zhao A. Huang H. Wei Z. Chen L. Qin S. Shao L. Xuan J. Feng G. et al.A novel heterozygous nonsense mutation of keratin 5 in a Chinese family with Dowling-Degos disease.J. Eur. Acad. Dermatol. Venereol. 2012; 26: 908-910Crossref PubMed Scopus (14) Google Scholar, 6Sprecher E. Indelman M. Khamaysi Z. Lugassy J. Petronius D. Bergman R. Galli-Galli disease is an acantholytic variant of Dowling-Degos disease.Br. J. Dermatol. 2007; 156: 572-574Crossref PubMed Scopus (40) Google Scholar, 7Liao H. Zhao Y. Baty D.U. McGrath J.A. Mellerio J.E. McLean W.H. A heterozygous frameshift mutation in the V1 domain of keratin 5 in a family with Dowling-Degos disease.J. Invest. Dermatol. 2007; 127: 298-300Crossref PubMed Scopus (51) Google Scholar, 8Arnold A.W. Kiritsi D. Happle R. Kohlhase J. Hausser I. Bruckner-Tuderman L. Has C. Itin P.H. Type 1 segmental Galli-Galli disease resulting from a previously unreported keratin 5 mutation.J. Invest. Dermatol. 2012; 132: 2100-2103Crossref PubMed Scopus (8) Google Scholar In subsequent years, we screened more than 40 individuals with DDD and found KRT5 mutations in fewer than 50%, with only 16 simplex and familial cases.9Hanneken S. Rütten A. Eigelshoven S. Braun-Falco M. Pasternack S.M. Ruzicka T. Nöthen M.M. Betz R.C. Kruse R. [Galli-Galli disease. Clinical and histopathological investigation using a case series of 18 patients].Hautarzt. 2011; 62: 842-851Crossref PubMed Scopus (12) Google Scholar, 10Hanneken S. Rütten A. Pasternack S.M. Eigelshoven S. El Shabrawi-Caelen L. Wenzel J. Braun-Falco M. Ruzicka T. Nöthen M.M. Kruse R. Betz R.C. Systematic mutation screening of KRT5 supports the hypothesis that Galli-Galli disease is a variant of Dowling-Degos disease.Br. J. Dermatol. 2010; 163: 197-200PubMed Google Scholar Thus, the causes of a large number of unsolved cases of DDD remain to be explained. In some individuals with DDD, who had originally been diagnosed with Galli-Galli disease (GGD), the additional histopathological feature of acantholysis was observed. The clinical presentation and the genetic backgrounds of these individuals indicated that GGD is a variant of DDD and not a distinct disease entity.6Sprecher E. Indelman M. Khamaysi Z. Lugassy J. Petronius D. Bergman R. Galli-Galli disease is an acantholytic variant of Dowling-Degos disease.Br. J. Dermatol. 2007; 156: 572-574Crossref PubMed Scopus (40) Google Scholar, 10Hanneken S. Rütten A. Pasternack S.M. Eigelshoven S. El Shabrawi-Caelen L. Wenzel J. Braun-Falco M. Ruzicka T. Nöthen M.M. Kruse R. Betz R.C. Systematic mutation screening of KRT5 supports the hypothesis that Galli-Galli disease is a variant of Dowling-Degos disease.Br. J. Dermatol. 2010; 163: 197-200PubMed Google Scholar, 11Schmieder A. Pasternack S.M. Krahl D. Betz R.C. Leverkus M. Galli-Galli disease is an acantholytic variant of Dowling-Degos disease: additional genetic evidence in a German family.J. Am. Acad. Dermatol. 2012; 66: e250-e251Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Another locus for DDD was identified in an affected Chinese family; this locus is on chromosome 17p13.3, but the responsible mutation has not been identified to date.12Li C.R. Xing Q.H. Li M. Qin W. Yue X.Z. Zhang X.J. Ma H.J. Wang D.G. Feng G.Y. Zhu W.Y. He L. A gene locus responsible for reticulate pigmented anomaly of the flexures maps to chromosome 17p13.3.J. Invest. Dermatol. 2006; 126: 1297-1301Crossref PubMed Scopus (28) Google Scholar Additionally, recent studies of two Chinese families with DDD led to the identification of mutations in POFUT1 (MIM 607491), which encodes protein O-fucosyltransferase 1 from the Notch pathway.13Li M. Cheng R. Liang J. Yan H. Zhang H. Yang L. Li C. Jiao Q. Lu Z. He J. et al.Mutations in POFUT1, Encoding Protein O-fucosyltransferase 1, Cause Generalized Dowling-Degos Disease.Am. J. Hum. Genet. 2013; 92: 895-903Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Here, we describe the identification of nine different mutations in POGLUT1 (RefSeq accession number NM_152305.2) from 13 unrelated individuals with DDD. POGLUT1 encodes protein O-glucosyltransferase 1 and is a part of the Notch signaling pathway.14Ma W. Du J. Chu Q. Wang Y. Liu L. Song M. Wang W. hCLP46 regulates U937 cell proliferation via Notch signaling pathway.Biochem. Biophys. Res. Commun. 2011; 408: 84-88Crossref PubMed Scopus (26) Google Scholar, 15Fernandez-Valdivia R. Takeuchi H. Samarghandi A. Lopez M. Leonardi J. Haltiwanger R.S. Jafar-Nejad H. Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi.Development. 2011; 138: 1925-1934Crossref PubMed Scopus (129) Google Scholar Further studies including immunohistochemistry, immunofluorescence, and immunoblotting supported the pathogenicity of the identified mutations. We used the whole-exome sequencing technology to identify other genetic causes of DDD. Five unrelated individuals with comparable DDD phenotypes were selected for sequencing under the assumption that any gene harboring rare variants in all individuals might be a DDD candidate. The age of onset in the five individuals varied between 18 and 53 years. These individuals were all described and characterized previously under a distinct clinical subtype suggested for DDD/Galli-Galli disease.9Hanneken S. Rütten A. Eigelshoven S. Braun-Falco M. Pasternack S.M. Ruzicka T. Nöthen M.M. Betz R.C. Kruse R. [Galli-Galli disease. Clinical and histopathological investigation using a case series of 18 patients].Hautarzt. 2011; 62: 842-851Crossref PubMed Scopus (12) Google Scholar Specifically, affected individuals presented with a disseminated pattern of brownish macular and lentiginous lesions on the extremities, trunk/back and neck without the typical domination of the flexural folds observed in classical DDD (Figures 1A–1F).9Hanneken S. Rütten A. Eigelshoven S. Braun-Falco M. Pasternack S.M. Ruzicka T. Nöthen M.M. Betz R.C. Kruse R. [Galli-Galli disease. Clinical and histopathological investigation using a case series of 18 patients].Hautarzt. 2011; 62: 842-851Crossref PubMed Scopus (12) Google Scholar Among the five selected individuals with DDD, three reported no family history of DDD. One male individual reported that his father was affected by skin abnormalities similar to his own (Figure 1G), and one female individual reported that her sister and probably her mother exhibited skin abnormalities similar to her own (Figure 1H). Ethical approval was obtained from the ethics committee of the Medical Faculty of the University of Düsseldorf and the participants provided written informed consent prior to blood sampling. The study was conducted in concordance with the Declaration of Helsinki Principles. DNA was extracted from peripheral blood leukocytes according to standard methods. For whole-exome sequencing, we fragmented 1 μg of DNA with sonication technology (Bioruptor, Diagenode, Liège, Belgium). The fragments were end-repaired and adaptor-ligated, including incorporation of sample index barcodes. After size selection, we subjected a pool of all 5 libraries to an enrichment process with the SeqCap EZ Human Exome Library version 2.0 kit (Roche NimbleGen). The final libraries were sequenced on an Illumina HiSeq 2000 sequencing instrument (Illumina) with a paired-end 2 × 100 bp protocol. This resulted in 6.2−7.3 Gb of mapped sequences (on average 6.8 Gb), a mean coverage of 68–80× (on average 75×) and 30× coverage of 82–86% (on average 84%) of the target sequences. The Varbank pipeline v.2.1 and interface were used for data analysis and filtering (unpublished data, H.T., J.A., and P.N.; see Table S1 available online). The data were filtered for high-quality rare (MAF < 0.005) autosomal variants and attention was focused on genes with the highest burden of functional variants in individuals with DDD. Sequencing data analysis identified a single gene, POGLUT1, which harbors heterozygous nonsense or splice site mutations in all five affected individuals (Figure 2). One of the investigated individuals showed a guanine-to-adenine transition at nucleotide position 11 leading to a stop codon (c.11G>A [p.Trp4∗]). We identified another nonsense mutation in two further individuals at nucleotide position 652 (c.652C>T [p.Arg218∗]; among them individual II:1, Figure 1G). In the remaining two individuals, we identified a mutation at a splice site (c.798-2A>C; among them individual II:1, Figure 1H). The splice site mutation was also identified in the sister of individual II:2 (Figure 1H; data not shown). All three variants were confirmed by Sanger sequencing using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and an ABI 3100 genetic analyzer (Applied Biosystems) (Figure 2B). In addition, we screened further individuals from our DDD cohort for mutations in POGLUT1. Primer sequences for amplification of POGLUT1 exons are listed in the supplemental information (Table S2). We identified c.11G>A in a single person, c.798-2A>C in a single person, and additional mutations in six individuals (Table 1; Figure 2A; Figure S1). One of these mutations, c.394C>T (p.Arg132∗), which leads to a premature stop codon, is reported in the 1000 Genomes database as the variant rs140695299 with a frequency of 1 in 1,092 individuals. Due to the low incidence frequency and the late age of onset of the disease, it is likely that this mutation and two further very rare (combined MAF < 0.0002) frameshift and splice site mutations (c.898 del1 [p.Phe300Serfs∗6] and c.1023-2A>C, respectively) reported in databases also lead to the hyperpigmentation disorder, and that DDD might be more common than reported. None of the other identified mutations were found in dbSNP137, ESP, or 1000 Genomes databases. Unfortunately, mostly as a result of the late age of onset of the disease, it was not possible to get blood samples from any parents of our individuals or any affected children. In total, we identified nine different heterozygous mutations in a total of 13 individuals with DDD, including nonsense, splice site, missense, insertion, and deletion mutations. We therefore suggest that POGLUT1 is a gene, mutations in which are responsible for DDD. Of note, one of the nonsense mutations is located at the very beginning of the protein (p.Trp4∗). Therefore, it is very likely that the mRNA transcript with this mutation is affected by nonsense-mediated mRNA decay making haploinsufficiency the most plausible mechanism for autosomal-dominant inheritance.Table 1POGLUT1 Mutations Identified in 13 Individuals with DDDMutationProtein AlterationNumber of IndividualsOrigin of the IndividualsReferencesc.11G>Ap.Trp4∗2Germany, DenmarkHanneken et al., 2011This studyc.205C>Tp.Arg69∗1SwitzerlandThis studyc.394C>Tp.Arg132∗1GermanyThis studyc.635C>Gp.Ser212∗1GermanyThis studyc.652C>Tp.Arg218∗2GermanyHanneken et al., 2011c.798-2A>C-3GermanyHanneken et al., 2011c.833_834insCArg279Profs∗31GermanyMauerer et al., 201042Mauerer A. Betz R.C. Pasternack S.M. Landthaler M. Hafner C. Generalized solar lentigines in a patient with a history of radon exposure.Dermatology. 2010; 221: 206-210Crossref PubMed Scopus (6) Google Scholarc.835C>Tp.Arg279Trp1GermanyThis studyc.1023_1025delGp.Gly342Glufs∗221GermanyThis study Open table in a new tab Of interest, the observation of prominent involvement of nonflexural areas in individuals with POGLUT1 mutations in comparison to the individuals with KRT5 mutations who present with typical domination of the flexural folds9Hanneken S. Rütten A. Eigelshoven S. Braun-Falco M. Pasternack S.M. Ruzicka T. Nöthen M.M. Betz R.C. Kruse R. [Galli-Galli disease. Clinical and histopathological investigation using a case series of 18 patients].Hautarzt. 2011; 62: 842-851Crossref PubMed Scopus (12) Google Scholar is suggestive of a correlation between the gene in which mutations are harbored and the DDD phenotype displayed by the affected individuals. POGLUT1 is located on chromosome 3q13.33 and has a 1.179 bp open reading frame consisting of 11 coding exons.16Teng Y. Liu Q. Ma J. Liu F. Han Z. Wang Y. Wang W. Cloning, expression and characterization of a novel human CAP10-like gene hCLP46 from CD34(+) stem/progenitor cells.Gene. 2006; 371: 7-15Crossref PubMed Scopus (26) Google Scholar POGLUT1 encodes the 392 amino acid protein POGLUT1, alternatively termed KTELC1, C3orf9, hCLP46, and Rumi, among others. POGLUT1 constitutes protein O-glucosyltransferase 1, which adds O-linked glucose to the epidermal growth factor-like (EGF) repeats of Notch receptors.15Fernandez-Valdivia R. Takeuchi H. Samarghandi A. Lopez M. Leonardi J. Haltiwanger R.S. Jafar-Nejad H. Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi.Development. 2011; 138: 1925-1934Crossref PubMed Scopus (129) Google Scholar, 17Takeuchi H. Fernández-Valdivia R.C. Caswell D.S. Nita-Lazar A. Rana N.A. Garner T.P. Weldeghiorghis T.K. Macnaughtan M.A. Jafar-Nejad H. Haltiwanger R.S. Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase.Proc. Natl. Acad. Sci. USA. 2011; 108: 16600-16605Crossref PubMed Scopus (65) Google Scholar, 18Acar M. Jafar-Nejad H. Takeuchi H. Rajan A. Ibrani D. Rana N.A. Pan H. Haltiwanger R.S. Bellen H.J. Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling.Cell. 2008; 132: 247-258Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar The mutations identified in our individuals with DDD are predicted to have a major impact on the translation or the structure of POGLUT1. POGLUT1 is orthologous to several other structurally well-characterized glucosyltransferases from viruses or bacteria. The effects of the different POGLUT1 mutations on the resultant protein were predicted on the basis of a homology model (Figures 3A, 3B, and 3E ; Figure S2). A model of POGLUT1 was generated with Modeler.19Sali A. Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints.J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10514) Google Scholar, 20Martí-Renom M.A. Stuart A.C. Fiser A. Sánchez R. Melo F. Sali A. Comparative protein structure modeling of genes and genomes.Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 291-325Crossref PubMed Scopus (2561) Google Scholar, 21Stuart P.E. Hüffmeier U. Nair R.P. Palla R. Tejasvi T. Schalkwijk J. Elder J.T. Reis A. Armour J.A. Association of β-defensin copy number and psoriasis in three cohorts of European origin.J. Invest. Dermatol. 2012; 132: 2407-2413Crossref PubMed Scopus (49) Google Scholar, 22Eswar N. Webb B. Marti-Renom M.A. Madhusudhan M.S. Eramian D. Shen M.Y. Pieper U. Sali A. Comparative protein structure modeling using Modeller.Curr Protoc Bioinformatics. 2006; Chapter 5 (Unit 5 6)PubMed Google Scholar Alignments of POGLUT1 with sequences of template structures were generated with the HHPred server.23Söding J. Biegert A. Lupas A.N. The HHpred interactive server for protein homology detection and structure prediction.Nucleic Acids Res. 2005; 33: W244-W248Crossref PubMed Scopus (2631) Google Scholar The model was manually adjusted with Coot24Emsley P. Cowtan K. Coot: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23229) Google Scholar and figures were generated with PyMol.25Schrödinger, L.L.C. (2010). The PyMOL Molecular Graphics System, Version 1.3r1. In.Google Scholar As in orthologous glucosyltransferases, POGLUT1 is composed of an N-terminal (residues 1–180) and a C-terminal (residues 181–392) domain that together form a large binding pocket for the substrate UDP-glucose at the domain interface (Figure 3A). The arrangement of the two domains is stabilized by a long C-terminal helix, whereby the C-terminal end of the helix is anchored in the N-terminal domain. The mutations causing a translation stop lead to truncated variants of POGLUT1 that lack major parts or essential residues resulting in the loss of the substrate binding site (Figure S2). The truncated form p.Arg132∗ is missing parts of the N-terminal domain as well as the entire C-terminal domain. The mutants p.Ser212∗ and p.Arg218∗, as well as p.Arg279Profs∗3, lack parts of the substrate-binding site essential for high-affinity binding of UDP-glucose. p.Gly342Glufs∗22 is missing in part the long C-terminal helix causing most likely a disassembly of the N- and C-terminal domains (Figure S2). We analyzed the effects of the splice site mutation c.798-2A>C by total RNA isolation from the mutation carrier individual followed by reverse transcription of total mRNA into cDNA and POGLUT1 sequencing. We showed that this mutation leads to abolishment of exon 9, which would result in loss of residues 267–322 in the translated protein (Figure S3). This large deletion will abolish correct folding of the C-terminal domain of POGLUT1. Moreover, a number of residues forming the substrate binding site will be missing, and therefore we anticipate that this mutant is also inactive. The p.Arg279Trp substitution (Figure 3C) leads to the replacement of a highly conserved arginine (Figure 3D) with the dissimilar amino acid tryptophan. The homology model revealed that Arg279 is particularly involved in binding the UDP-glucose substrate. In the orthologous glucosyltransferase of T4 phage, the guanidinium group of the corresponding arginine residue forms two salt bridges with the diphosphate moiety of UDP-glucose and is required to bind the substrate with high affinity.26Moréra S. Imberty A. Aschke-Sonnenborn U. Rüger W. Freemont P.S. T4 phage beta-glucosyltransferase: substrate binding and proposed catalytic mechanism.J. Mol. Biol. 1999; 292: 717-730Crossref PubMed Scopus (99) Google Scholar, 27Moréra S. Larivière L. Kurzeck J. Aschke-Sonnenborn U. Freemont P.S. Janin J. Rüger W. High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding.J. Mol. Biol. 2001; 311: 569-577Crossref PubMed Scopus (62) Google Scholar In the homology model, Arg279 can adopt a similar conformation to bind UDP-glucose (Figure 3B). Substitution by tryptophan disrupts the interaction with the substrate and, moreover, the bulky side chain of tryptophan might hinder access of the substrate to the binding site (Figure 3E). To analyze the localization pattern of POGLUT1, we investigated skin biopsies from healthy and affected individuals by using immunohistochemistry. Sections were prepared from formalin-fixed, paraffin-embedded skin biopsies obtained by plastic surgery from four individuals with DDD and different POGLUT1 mutations (nonsense and splice site) and from four healthy controls with normal skin. Standard H&E and periodic acid-Schiff staining was performed for diagnostic purposes. POGLUT1 localization was analyzed using polyclonal anti-POGLUT1 antibody. Results were evaluated on blinded specimens by an experienced dermatopathologist (J.W.) as described previously.28Wenzel J. Wörenkämper E. Freutel S. Henze S. Haller O. Bieber T. Tüting T. Enhanced type I interferon signalling promotes Th1-biased inflammation in cutaneous lupus erythematosus.J. Pathol. 2005; 205: 435-442Crossref PubMed Scopus (188) Google Scholar Histologically, skin lesions from individuals with DDD showed a digitiform reteacanthosis with pronounced hyperpigmentation at the tips of the rete ridges, some small horn cysts, minor acantholysis, and focal hypergranulosis (Figure S4). In immunohistology, we found POGLUT1 to be prominently present in the epidermis of healthy controls, especially in the upper parts (stratum spinosum and stratum granulosum, Figure 4A). The strong POGLUT1 staining in the matured parts of the epidermis might be indicative that POGLUT1 is important for the correct differentiation of the epidermal layer and might emphasize an important role for POGLUT1 in the development of the epidermis. On the other hand, POGLUT1 staining was about 50% weaker in lesional skin of individuals with DDD in comparison to healthy controls (mean staining intensity, 1.75 ± 0.25 SEM versus 0.75 ± 0.32 SEM, p < 0.05, Mann-Whitney U-test) (Figure 4A; Figure S5). This observation could be due to only the WT POGLUT1 being detected by the antibody and not the truncated forms. This would be in accordance with the affected individuals having only one copy of the gene encoding for the WT protein. We next examined whether the mutated gene variants produce full-length, truncated, or no POGLUT1. For this purpose, POGLUT1, WT, and two of the identified mutants (p.Arg218∗ and p.Arg279Trp) were fused to N- and C-terminal Strep/FLAG Tandem Affinity-tags (N-TAP and C-TAP constructs, respectively) and expressed in HEK293T cells (European Collection of Cell Cultures [ECACC]) and analyzed by immunoblotting. Primers used for cloning and mutagenesis are listed in the supplemental information (Tables S3 and S4). Immunoblot analysis showed that the WT construct led to translation of a protein of about 50 kDa in size, which is in accordance with previous reports.16Teng Y. Liu Q. Ma J. Liu F. Han Z. Wang Y. Wang W. Cloning, expression and characterization of a novel human CAP10-like gene hCLP46 from CD34(+) stem/progenitor cells.Gene. 2006; 371: 7-15Crossref PubMed Scopus (26) Google Scholar While the missense mutation did not alter the molecular weight, the nonsense mutation resulted in a truncated protein of about 30 kDa in size (Figure 4B). There were no significant differences in the molecular weights between the N-TAP and C-TAP tagged proteins (Figure S6). The immunoblot labeled with anti-POGLUT1 antibody confirmed the translation of the targeted proteins (Figure 4B; Figure S6). To determine the subcellular localization of WT and mutant POGLUT1, we performed immunofluorescence analysis with transiently transfected HEK293T cells. The confocal microscopy analyses revealed the colocalization of the WT POGLUT1 with the endoplasmic reticulum (ER) for both the N- and C-TAP constructs (Figure 4C; Figure S7) as previously reported in COS7 cells.16Teng Y. Liu Q. Ma J. Liu F. Han Z. Wang Y. Wang W. Cloning, expression and characterization of a novel human CAP10-like gene hCLP46 from CD34(+) stem/progenitor cells.Gene. 2006; 371: 7-15Crossref PubMed Scopus (26) Google Scholar No significant difference was observed in the subcellular localization of the WT protein and the protein with the p.Arg279Trp substitution in either of the constructs (Figure 4C; Figure S7). However, compared to the WT proteins, the truncated N-TAP-POGLUT1 appeared to form more aggregates, which coincided with an impaired colocalization with the ER (Figure 4C). This can be explained by the lack of the C-terminal tetrapeptide sequence KTEL, which is known to be important for the retention of POGLUT1 in the ER.16Teng Y. Liu Q. Ma J. Liu F. Han Z. Wang Y. Wang W. Cloning, expression and characterization of a novel human CAP10-like gene hCLP46 from CD34(+) stem/progenitor cells.Gene. 2006; 371: 7-15Crossref PubMed Scopus (26) Google Scholar In addition, the stabilizing effect of the C-terminal helix as shown by homology modeling of POGLUT1 (Figure 3A) might influence the protein conformation and thus its subcellular localization. In the C-TAP constructs, however, we did not observe any significant difference in the localization of the WT and the truncated proteins (Figure S7). The difference between the two constructs can be attributed to the position of the tag. In the C-TAP constructs the amino acid sequence in the vicinity of the tag is different between the WT and the truncated forms, whereas it is the same in the N-TAP constructs. POGLUT1 is part of the Notch signaling pathway, which is important for cell fate and tissue formation during embryogenesis and, in adulthood, for differentiation and stem cell maintenance. Mutations have been found in various genes of the Notch signaling pathway, including those encoding receptors and ligands, an
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