Molecular mechanism of CHRDL1-mediated X-linked megalocornea in humans and in Xenopus model

Chordin-Like 1 (CHRDL1) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC—with presumably disturbed cornea growth and differentiation—contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injurie

Identification of FOXP1 Deletions in Three Unrelated Patients with Mental Retardation and Significant Speech and Language Deficits

Mental retardation affects 2-3% of the population and shows a high heritability. Neurodevelopmental disorders that include pronounced impairment in language and speech skills occur less frequently. For most cases, the molecular basis of mental retardation with or without speech and language disorder is unknown due to the heterogeneity of underlying genetic factors. We have used molecular karyotyping on 1523 patients with mental retardation to detect copy number variations (CNVs) including deletions or duplications. These studies revealed three heterozygous overlapping deletions solely affecting the forkhead box P1 (FOXP1) gene. All three patients had moderate mental retardation and significant language and speech deficits. Since our results are consistent with a de novo occurrence of these deletions, we considered them as causal although we detected a single large deletion including FOXP1 and additional genes in 4104 ancestrally matched controls. These findings are of interest with regard to the structural and functional relationship between FOXP1 and FOXP2. Mutations in FOXP2 have been previously related to monogenic cases of developmental verbal dyspraxia. Both FOXP1 and FOXP2 are expressed in songbird and human brain regions that are important for the developmental processes that culminate in speech and language. ©2010 Wiley-Liss, Inc

Biochemical engineering of HL-60 cells: integrin expression and integrin dependent adhesion

Titel Inhaltsverzeichnis 1 1\. Einleitung 4 2\. Zielsetzung 20 3\. Ergebnisse Teil 1 21 4\. Ergebnisse Teil 2 29 5\. Diskussion 38 6\. Zusammenfassung 52 7\. Material und Methoden 53 8\. Literaturverzeichnis 71 Anhang, Danksagung, Lebenslauf, PublikationBiochemical-Engineering ist ein effizientes Modifikationsverfahren für Glycoproteine der zellulären Plasmamembran. Es soll dazu dienen, strukturabhängige Funktionen von N- und O-Glycanen zu untersuchen. Ein wichtiges Ziel dieses Verfahrens sind Sialinsäuren (Synonym: Neuraminsäuren). Sie zeichnen sich als terminalgebundene Glycankomponenten durch ihre strukturelle Vielfalt und ihre dadurch differenzierten Funktionen bei der Embryonalentwicklung, Immunabwehr und Krebsentstehung aus. Diese Arbeit sollte zeigen, ob sich durch spezifische Modifikation der N-Acyl-Seitenkette von Sialinsäuren mittels des unphysiologischen Sialinsäure-Vorläufers N-Propanoyl-D-mannosamin (ManNProp) die ß1-Integrin-abhängige Adhäsion der humanen Leukämiezellinie HL-60 auf Fibronektinmatrix modulieren läßt. Es konnte gezeigt werden, daß HL-60-Zellen durch ManNProp langfristig verstärkt ß1-Integrin-abhängig auf Fibronektinmatrix adhärieren. Dies ging mit vermehrter ß1-Integrinexpression auf Proteinebene einher, zwar unabhängig von der Zelloberflächenexpression, aber im Zusammenhang mit verändertem Umbau und Clusterformierung von ß1-Integrinen, die adhäsionstypische morphologische Zellspreizung erzeugten. Zusätzlich konnte gezeigt werden, daß durch Hemmung der Signalmolekülfamilie Proteinkinase C (PKC) die adhäsionsinduzierende Wirkung von ManNProp auf die Leukämiezellinie HL-60 reversibel ist. Die Ergebnisse dieser Arbeit unterstreichen die Bedeutung strukturabhängiger Einflüsse von Glycanketten, inbesondere ihrer terminalgebundenen Sialinsäuren, auf Glycoprotein-gesteuerte Mechanismen wie der Zelladhäsion. Außerdem wird gezeigt wie durch spezifisch veränderte Analoga solche Basisfunktionen der Zelle moduliert werden können. Dies könnte beispielsweise bei der Regulation von Leukozyten-Homing im Rahmen entzündlicher Prozesse von Bedeutung sein.Biochemical engineering is an efficient modification procedure for glycoproteins of the plasmamembrane to reveal functional relevance of N- and O-glycans especially of terminal bound sialic acids (synonym: neuraminic acids). Sialic acids have due to their structure diversity distinct implications in embryogenesis, immunresponse und cancerogenesis. The aim of this experimental work was to messure the effect of specific N-Acyl-side chain elongation of the sialic acids with the unphysiological precursor N-propanoyl-D-mannosamine (ManNProp) on integrin-dependent adhesion of the human leucemia cell line HL-60 upon fibronectin matrix. This work shows that prolonged incubation of HL-60 cells with ManNProp induces their ß1-integrin- dependent adherence on fibronectin matrix going along with increased expression of ß1-integrins on protein level. Although there was no increased ß1-integrin expression on the cell surface, the turnover and cluster formation of ß1-integrins was induced, latter responsible for cellspreading. Also it has been shown that inhibition of the Proteinkinase C (PKC) family was able to reduce the ManNProp effect on adhesion of HL-60 cells upon fibronectin matrix. These results emphasize the important role of structure dependent implications of glycans especially their terminal sialic acids on glycoprotein regulted processes like cell adhesion. Also it is shown how specific modified analogues modulate basic functions of the cell. This could have clinical relevance for example in the regulation of leukocyte homing during inflammation

Rmnd5 is part of an ubiquitin ligase complex.

<p>(A) Glycerol step gradient of <i>Xenopus laevis</i> NF stage 36 embryo lysates. Molecular mass (MW) standard: albumin (67 kDa), fraction 1, 2; LDH (140 kDa), fraction 4; catalase (232 kDa), fraction 6,7. Western blot analysis with α-RMND5A (Rmnd5; upper panel) (1:1000) and α-ARMC8 (lower panel) (1:1000). (B) <i>In vitro</i> polyubiquitination assay with recombinant <i>Xenopus</i> Rmnd5 and Rmnd5-C354S (lane 3, 4). Reactions are performed in the presence (+) or absence (-) of E1 (lane 1), E2 (lane 2) and purified Rmnd5 protein. HDM2 is used as a positive control (lane 5). Polyubiquitination (Poly-Ub) is detected with α-HA and α-RMND5A as control.</p

<i>rmnd5</i> is expressed during early embryonic development.

<p>(A) Temporal RT-PCR analysis of <i>rmnd5</i> expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of <i>rmnd5</i> expression. Whole mount <i>in situ</i> hybridisation (Wmish) of wild type <i>Xenopus laevis</i> embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) <i>rmnd5</i> transcript in the animal pole (top), NF-stage 12 (panel d) <i>rmnd5</i> transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).</p

The CTLH complex functions during early embryonic neurogenesis.

<p>(A) <i>rmnd5</i>-mo injected embryos were used for <i>in situ</i> hybridisation with indicated marker probes; pax6 (upper lane), n-tubulin (middle lane), k20/en22/rx1/c-actin, emx1.2, nkx2.1 (bottom lanes). Abbreviations: IS, injected side; NIS, non-injected side. Quantitative representation of phenotypes are presented as a bar graph (percent embryos with phenotype to total amount (%); black, phenotype; grey, no phenotype); n = number of independent experiments, N = number of injected embryos analysed for respective marker, *P ≤0.05, ***≤0.001 (Chi Square test). (B) As (A) with <i>sox2</i> as probe. (C) Xenopus embryos co-injected with <i>rmnd5</i> morpholino (2.5 pmol/embryo) and synthetic capped RNA (100 pg/embryo) were used for <i>in situ</i> hybridisation and quantified as shown in A.</p

<i>Xenopus laevis</i> Rmnd5 protein is structurally and functionally related to human RMND5A.

<p>(A) Phylogenetic tree of Rmnd5 orthologs. The taxonomic tree of representative eukaryotic species rendered by Phylogeny.fr software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120342#pone.0120342.ref020" target="_blank">20</a>]. Respective Gid2/Rmnd5 sequences obtained from NCBI with indicated accession numbers (<i>Saccharomyces cerevisiae</i> [NP_010541.3], <i>Candida albicans</i> [XP_712238.1], <i>Aspergillus niger</i> [XP_001388791.2], <i>Caenorhabditis elegans</i> [NP_508444.1], <i>Arabidopsis thaliana</i> [NP_196525.1], <i>Drosophila melanogaster</i> [NP_611536.3], <i>Xenopus laevis</i> [NP_001086276.1], <i>Falco peregrinus</i> [XP_005229906.1], <i>Gallus gallus</i> [XP_004936301.1] <i>Homo sapiens</i> [NP_073617.1; NP_073599.2], <i>Ornithorhynchus anatinus</i> [XP_007670084.1; XP_001515875.2], <i>Sarcophilus harrisii</i> [XP_003758697.1; XP_003756956.1], <i>Canis lupus familiaris</i> [XP_852129.1; XP_531873.2], <i>Mus musculus</i> [NP_077250.2; NP_079622.1], <i>Rattus norvegicus</i> [XP_232051.4; NP_001017473.1]); homolog A (blue), homolog B (red). (B) Sequence alignment of <i>Xenopus laevis</i> Rmnd5 (top), <i>Homo sapiens</i> RMND5A (middle) and RMND5B (bottom). Identical residues (red), similar residues (blue), others (black). Identities (%): <i>Xenopus laevis</i> Rmnd5 to human RMND5A (94%), to human RMND5B (70%). (C) Localization of <i>Homo sapiens</i> RMND5A (RMND5a, middle panel), RMND5B (RMND5b, bottom panel) and <i>Xenopus laevis</i> Rmnd5 (Rmnd5, top panel) in HEK293 cells. GFP signal (left column), DAPI signal (middle column), merged signals (right column).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Primers used in this study.

<p>Primers used in this study.</p

RMND5 from <i>Xenopus laevis</i> Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

<div><p>In <i>Saccharomyces cerevisiae</i> the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of <i>rmnd5</i> is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that <i>Xenopus laevis</i> Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.</p></div