The impact of sequence variation on Acid Sphingomyelinase activity in the context of Major Depressive Disorder
Der Einfluss von Sequenzvariationen auf die Aktivität der Sauren Sphingomyelinase im Kontext der Majoren Depression
Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von Cosima Rhein aus Fürth
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 23. Mai 2011
Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Uwe Sonnewald
Zweitberichterstatter: Prof. Dr. Johannes Kornhuber
ἔνθ ᾽ αὖτ᾽ ἄλλ ᾽ ἐνόησ ᾽ Ἑλένη ι ὸς ἐκγεγαυ ῖα αὐτίκ ᾽ ἄρ᾽ εἰς οἶνον βάλε φάρ ακον , ἔνθεν ἔπινον , νηπενθές τ᾽ ἄχολόν τε , κακ ῶν ἐπίληθον ἁπάντων .
Da entsann die zeusentsprossene Helena andres. Und sie warf in den Wein, von welchem sie tranken, ein Mittel gegen Kummer und Groll und aller Übel Gedächtnis.
Homer, Odyssee
Danksagung
Herrn Professor Kornhuber danke ich dafür, dass ich meine Doktorarbeit an der Psychiatrischen und Psychotherapeutischen Klinik durchführen und die ausgezeichnete Infrastruktur des Labors für molekulare Neurobiologie nutzen durfte. Das interdisziplinäre Arbeiten an der Klinik hat mir interessante Einblicke in das Feld der klinischen Neurowissenschaften ermöglicht. Eine bereichernde Erfahrung war es auch, an verschiedenen internationalen Kongressen teilnehmen zu dürfen – herzlichen Dank dafür.
Großer Dank gilt Herrn Professor Sonnewald für die Übernahme der Begutachtung dieser Arbeit.
Bei Herrn Dr. Martin Reichel bedanke ich mich für die inhaltliche Betreuung dieser Arbeit, seine Diskussionsbereitschaft und sein Vertrauen in mein experimentelles Arbeiten. Besonders danke ich ihm für die verlässliche Zusammenarbeit. Herrn Dr. Philipp Tripal danke ich für die praktische Betreuung dieser Arbeit, seine große Hilfsbereitschaft und die wertvolle Unterstützung. Ich danke ihm sehr, dass er mir viele Methoden nahe gebracht und mich für das experimentelle Arbeiten begeistert hat.
Ein großes Dankeschön geht an Frau Alice Konrad für die exzellente experimentelle Unterstützung im letzten Jahr meiner Doktorarbeit. Ihre perfekte und weitsichtige Arbeitsweise in sämtlichen Methoden des Labors machte es zum Vergnügen, mit ihr zusammen zu arbeiten. Frau Michaela Schäfer danke ich sehr für die hervorragende Unterstützung im Bereich der Zellkultur. Der hohe Standard ihrer Arbeitsweise ermöglichte erst eine Vielzahl an Experimenten mit verschiedensten Zelllinien. Bei Frau Sabine Müller bedanke ich mich für die ausgezeichnete Unterstützung bei den unzähligen Schritten des Klonierens und ihre äußerst zuverlässige Arbeitsweise.
Frau Dr. Christiane Mühle danke ich für TLC-Messungen, die stets interessanten Ideen bezüglich des Laboralltags und die Versorgung mit Werbematerial und Sprossen. Ein Dankeschön gilt Herrn Stefan Hofmann für TLC-Messungen und seine unterhaltsamen Gedankenexperimente über verschiedenste Themen. Bei Frau Dr. Angela Seebahn bedanke ich mich für die unkomplizierte und erfolgreiche Kooperation bezüglich der Massenspektrometrie. Frau Dr. Rotter-Neubert und Herrn Dr. Bernd Lenz danke ich für die gute Zusammenarbeit und ihre Bereitschaft, meine Experimente jederzeit durch Blutentnahmen zu unterstützen. Bei den Mitarbeitern der Abteilung für molekulare Neurologie bedanke ich mich für die fruchtbare Zusammenarbeit und die kollegiale Nachbarschaft. Allen aktuellen und bereits ausgeschiedenen Kollegen der molekularen Neurobiologie sage ich Dankeschön dafür, dass sie die Zeit während meiner Doktorarbeit mitgestaltet und meist bereichert haben.
Meinem Molmädels-Stammtisch danke ich sehr für die fachliche und mentale Unterstützung. Herrn ORR Bernd W. Göppner danke ich für die umfassende Unterstützung, sein Verständnis für alle Widrigkeiten des Forscherlebens und seine Fähigkeit, mich stets zu motivieren. Von Herzen danke ich meinen Eltern und Schwestern für den unerschütterlichen Beistand in allen Lebenslagen.
Contents
Contents
List of figures ...... VIII List of tables ...... IX List of abbreviations...... X
1. Zusammenfassung...... 12 Summary ...... 13
2. Introduction ...... 14 2.1 Lysosomal sphingolipid disorders of the brain ...... 14 2.1.1 Sphingolipids play diverse roles in cellular processes...... 15 2.1.1.1 Sphingolipids: Functional diversity by structural diversity...... 15 2.1.1.2 Roles of sphingolipids for the CNS ...... 16 2.1.1.3 The lipid messenger ceramide ...... 16 2.1.2 Molecular defects of lysosomal enzymes ...... 18 2.1.2.1 Primary defects ...... 19 2.1.2.2 Posttranslational defects...... 20 2.1.2.3 Defective trafficking ...... 20 2.2 Acid sphingomyelinase in health and disease...... 21 2.2.1 Acid Sphingomyelinase – the lysosomal enzyme for sphingomyelin breakdown ...... 21 2.2.1.1 Genomic organization and transcription...... 22 2.2.1.2 Sequence variations of Acid Sphingomyelinase...... 23 2.2.1.3 Properties of the Acid Sphingomyelinase protein ...... 23 2.2.2 Regulation of Acid Sphingomyelinase activity ...... 25 2.2.2.1 Transcriptional level ...... 25 2.2.2.2 Posttranscriptional level...... 26 2.2.2.3 Posttranslational modifications...... 26 2.2.2.4 Regulation via degradation ...... 26 2.2.3 Acid Sphingomyelinase and pathologic context...... 27 2.2.3.1 Decreased activity of Acid Sphingomyelinase ...... 27 2.2.3.2 Increased activity of Acid Sphingomyelinase...... 28 2.3 The role of Acid Sphingomyelinase in Major Depressive Disorder ...... 28 2.3.1 Results of the pilot study...... 29 2.3.2 Sequence variations of Acid Sphingomyelinase in depressed patients ...... 30 2.4 Aims of the study...... 30
3. Material and Methods...... 32 3.1 Material...... 32 3.1.1 Enzymes...... 32 3.1.2 Antibodies...... 32 3.1.2.1 Primary antibodies ...... 32 3.1.2.2 Secondary antibodies ...... 33 3.1.3 Vectors and plasmids ...... 33 3.1.3.1 Vectors ...... 33
V Contents
3.1.3.2 Plasmids...... 33 3.1.4 Biological material...... 35 3.1.4.1 Bacteria ...... 35 3.1.4.2 Eucaryotic cells...... 35 3.1.5 Buffers and solutions ...... 35 3.2 Methods...... 39 3.2.1 Cell-biological methods...... 39 3.2.1.1 Cell culture...... 39 3.2.1.2 Transient expression of ASM ...... 40 3.2.1.3 Immunocytochemistry ...... 40 3.2.1.4 Preparation of whole cell extracts...... 41 3.2.2 DNA technology...... 41 3.2.2.1 PCR for cloning approach...... 41 3.2.2.2 Sub-cloning of ASM into pmCherry-N1 expression vector ...... 42 3.2.2.3 Site-directed mutagenesis ...... 43 3.2.2.4 PCR for detection of splice variants ...... 43 3.2.2.5 Agarose gel electrophoresis ...... 44 3.2.2.6 Cloning of ASM into FLAG-N2 expression vector...... 45 3.2.2.7 Transformation of bacteria with DNA...... 46 3.2.2.8 Isolation of plasmid DNA...... 47 3.2.2.9 Sequencing of cDNA ...... 47 3.2.3 RNA isolation and cDNA synthesis ...... 48 3.2.3.1 RNA isolation ...... 48 3.2.3.2 Synthesis of cDNA ...... 49 3.2.4 Protein-biochemical analyses...... 49 3.2.4.1 Determination of protein concentration...... 49 3.2.4.2 SDS-PAGE and Western blot analysis ...... 49 3.2.4.3 In vitro determination of ASM activity ...... 51 3.2.4.4 MALDI-TOF MS analysis of cellular ceramide and sphingomyelin levels ...... 52 3.2.5 In silico analyses and statistics ...... 52
4. Results...... 53 4.1 Impact of DNA sequence variations on Acid Sphingomyelinase activity ...... 53 4.1.1 Expression of recombinant ASM variants ...... 55 4.1.2 Characterization of ASM variants used for model system ...... 56 4.1.2.1 Catalytic activity of ASM model system variants ...... 57 4.1.2.2 Localization of ASM model system variants...... 58 4.1.3 Characterization of ASM variant p.P325A...... 60 4.1.3.1 ASM variant p.P325A is catalytically inactive...... 61 4.1.3.2 ASM variant p.P325A localizes in lysosomes...... 61 4.1.4 Characterization of ASM variant p.A487V ...... 63 4.1.4.1 Catalytic activity of ASM variant p.A487V is equivalent to wild type..63 4.1.4.2 ASM variant p.A487V localizes in lysosomes ...... 64 4.1.5 Characterization of ASM variant p.G506R ...... 65 4.1.5.1 ASM variation p.G506R impacts catalytic activity...... 65 4.1.5.2 ASM variation p.G506R impacts localization...... 66 4.1.6 Summary: Characterization of ASM variants...... 69 4.2 Impact of alternative splicing on Acid Sphingomyelinase activity...... 70 4.2.1 Structure of new ASM splice variants ...... 73 4.2.2 New splicing motifs are conserved in primates ...... 74
VI Contents
4.2.3 Biochemical characterization of new ASM splice variants ...... 74 4.2.3.1 New ASM splice variants are expressed in a cell culture model...... 77 4.2.3.2 New ASM splice variants are catalytically inactive in vitro ...... 77 4.2.3.3 New ASM splice variants are catalytically inactive in vivo ...... 78 4.2.4 New ASM splice variants localize within different subcellular compartments..79 4.2.5 New ASM splice variants show differential expression patterns in human blood cells ...... 82 4.2.5.1 New ASM splice variants are detected specifically by RT-PCR...... 82 4.2.5.2 New ASM splice variants are differentially expressed in healthy volunteers...... 83 4.2.5.3 New ASM splice variants are differentially expressed in depressed patients before and after antidepressant therapy...... 84 4.2.6 New splice variant ASM-5 exerts a dominant-negative effect on full-length Acid Sphingomyelinase...... 86 4.2.6.1 Upon serum starvation ASM-5 localizes within lysosomes ...... 86 4.2.6.2 ASM-5 is a dominant-negative ASM isoform...... 88
5. Discussion ...... 90 5.1 DNA sequence variations impact Acid Sphingomyelinase activity ...... 90 5.1.1 The C-terminal domain harbours essential parts for activation and secretion of Acid Sphingomyelinase ...... 91 5.1.2 Processing of Acid Sphingomyelinase is essential for its regulation in pathologic context...... 92 5.2 Alternative splicing impacts Acid Sphingomyelinase activity ...... 93 5.2.1 Alternative splicing of Acid Sphingomyelinase could predominate in brain and blood cells ...... 94 5.2.2 Acid Sphingomyelinase displays a defined set of conserved splicing motifs ....94 5.2.3 Acid Sphingomyelinase activity is regulated by alternative splicing ...... 96
6. Bibliography...... 98 7. Appendix ...... 107 7.1 Chemicals...... 107 7.2 Technical device ...... 108 7.3 Auxiliary material...... 108
VII List of figures
List of figures
Fig. 2.1 The ‘ceramide / sphingosine-1-phosphate rheostat’ concept...... 17 Fig. 2.2 ASM is subject to alternative splicing events...... 23 Fig. 2.3 ASM is composed of several domains...... 24 Fig. 4.1 Position of ASM variations investigated in this study...... 53 Fig. 4.2 Cloning approach for the generation of recombinant ASM constructs containing different ASM variations and FLAG- or Cherry-epitope ...... 54 Fig. 4.3 All investigated ASM variants are translated into protein and display predicted sizes...... 55 Fig. 4.4 ASM variants were transiently expressed to a different extent ...... 56 Fig. 4.5 ASM model system variants display predicted enzymatic activity levels...... 58 Fig. 4.6 Subcellular localization patterns of ASM model system variants ASM wild type and ASM variants p.S508A and p.M381I...... 59 Fig. 4.7 Co-localization of ASM model system variants with subcellular organelles...... 60 Fig. 4.8 ASM variant p.P325A does not exert catalytic activity compared to ASM wild type...... 61 Fig. 4.9 ASM variant p.P325A localizes in a dotted subcellular pattern ...... 62 Fig. 4.10 ASM variant p.P325A is localized within lysosomes...... 62 Fig. 4.11 Enzymatic activities of ASM variant p.A487V and ASM wild type reach similar levels ...... 63 Fig. 4.12 ASM variant p.A487V localizes in a dotted subcellular pattern ...... 64 Fig. 4.13 ASM variant p.A487V localizes within lysosomes ...... 64 Fig. 4.14 ASM variation p.G506R causes a decrease in enzymatic activity...... 66 Fig. 4.15 Subcellular localization patterns of ASM variants containing the variation p.G506R...... 67 Fig. 4.16 Co-localization of ASM variants containing variation p.G506R with subcellular organelles...... 68 Fig. 4.17 Coding sequences of full-length ASM and new alternatively-spliced transcripts differ locally...... 72 Fig. 4.18 Schematic structure of new splice variants ASM-5, -6 and -7 ...... 73 Fig. 4.19 Splicing patterns of novel transcripts ASM-6 and -7 are conserved in primates...74 Fig. 4.20 Novel variants ASM-5 to -7 differ regarding their putative protein sequence and protein domain structure ...... 76 Fig. 4.21 Expression of ASM-FLAG constructs in HeLa cells ...... 77 Fig. 4.22 Novel variants ASM -5 to -7 are catalytically inactive...... 78 Fig. 4.23 New ASM splice variants modulate ceramide to sphingomyelin ratios...... 79 Fig. 4.24 Subcellular localization patterns of ASM-1 and new ASM splice variants...... 80 Fig. 4.25 Co-localization of new ASM splice variants with subcellular organelles...... 81 Fig. 4.26 Designed oligonucleotides serve for specific amplification of new ASM splice variants...... 83 Fig. 4.27 Expression patterns of ASM splice variants vary in human blood cells...... 84 Fig. 4.28 Expression patterns of ASM splice variants vary in blood cells of depressed patients before and after administering of antidepressants...... 85 Fig. 4.29 Subcellular localization pattern of ASM-5 upon serum starvation...... 87 Fig. 4.30 ASM-5 localizes within lysosomes upon serum starvation ...... 87 Fig. 4.31 ASM-5 exerts a dominant-negative effect on ASM-1 upon serum starvation...... 89
VIII List of tables
List of tables
Tab. 3.1 Primary antibodies in cotext of their application...... 32 Tab. 3.2 Secondary antibodies in cotext of their application...... 33 Tab. 3.3 Vectors used in this study ...... 33 Tab. 3.4 Plasmids used in this study ...... 34 Tab. 3.5 Oligonucleotides used for cloning of ASM into pSC-B vector and FLAG-N2 expression vector ...... 42 Tab. 3.6 Oligonucleotides used for cloning of ASM from FLAG-N2 vector into pmCherry-N1 vector...... 42 Tab. 3.7 Oligonucleotides used for site-directed mutagenesis of ASM wild type-FLAG construct to generate different ASM variants ...... 43 Tab. 3.8 Oligonucleotides used for amplification of ASM splice variants...... 44 Tab. 3.9 Oligonucleotides used for sequencing of ASM plasmids...... 47 Tab. 4.1 ASM variants display differential properties...... 69
IX List of abbreviations
List of abbreviations
AA amino acid AC Acid Ceramidase APS ammonium persulfate ASM Acid Sphingomyelinase BES 2-hydroxyethyl-2-aminoethanesulfonic acid bp base pairs BSA bovine serum albumine c centi (10 -2) °C degree Celsius C-terminal carboxy-terminal CNS central nervous system cDNA coding DNA DMEM Dulbecco’s Modified Eagle’s Medium DMSO dimethylsulfoxide DMF 2,5-dimethylfurane DNA deoxyribonucleic acid dNTP deoxyribonucleoside-5’-triphosphate DTT dithiotreitol EC enzyme classification number ECL enhanced chemiluminescence E. coli Escherichia coli EDTA ethylenediaminetetraacetate ER endoplasmic reticulum FCS fetal calf serum Fig. figure fw forward g grams g gravitation constant GAPDH glycerinaldehyd-3-phosphat-dehydrogenase GFP green fluorescent protein h hour(s) H4 neuroglioma cell line H2Obidest aqua bidestillata HPRT hypoxanthine-phosphoribosyl transferase kb kilo base pairs kDa kilo daltons l litre LAMP1 lysosomal-associated membrane protein 1 LB Luria Bertani Medium LPS lipopolysaccharide M molar m milli (10-3) µ micro (10 -6) MDD major depressive disorder mRNA messenger ribonucleic acid min minute(s) MALDI-TOF MS matrix assisted laser desorption/ionisation time of flight mass spectrometry MCS multiple cloning site
X List of abbreviations ng nanogram nmol nanomole NPD Niemann-Pick disease N-terminal amino-terminal PAGE polyacrylamide gel electrophoresis PBMC peripheral mononuclear cell PBS phosphate buffered saline PCR polymerase chain reaction PDIA4 protein disulfide isomerase family A, member 4 pH -log H+ concentration PVDF polyvinyldifluoride RNA ribonucleic acid rev reverse rpm rounds per minute RT room temperature RT-PCR reverse transcription-PCR SAP saposin domain SDS sodium dodecyl sulphate sec second(s) SMPD1 sphingomyelin phosphodiesterase SNP single nucleotide polymorphism SOC medium super optimal broth medium containing glucose Tab. table TEMED N,N,N’,N’-tetramethylethylenediamine Tris Tris-hydroxymethyl-aminomethane Tween-20 polysorbate 20 U Unit UV ultraviolett V Volt Vmax maximum velocity of enzymes YT yeast extract and tryptone
XI 1. Zusammenfassung
1. Zusammenfassung
In einer Pilotstudie, die im Jahr 2005 von Kornhuber und Kollegen veröffentlicht wurde, war gezeigt worden, dass die Aktivität der sauren Sphingomyelinase (ASM) bei Patienten mit schwerer Depression im Vergleich zu einer gesunden Kontrollgruppe deutlich erhöht war. In der vorliegenden Arbeit wurde der mögliche Einfluss von ASM-Sequenzvariationen auf die Enzymaktivität als molekulare Ursache dieser Assoziation untersucht. Es wurde sowohl die Bedeutung von allgemein auftretenden DNA-Sequenzvariationen, die auch in einem Kollektiv depressiver Patienten identifiziert worden waren geprüft, als auch von RNA- Sequenzvariationen, um den Einfluss von alternativem Spleißen auf die zelluläre ASM- Aktivität zu beleuchten. Drei nicht-synonyme Sequenzvariationen im Bereich des maturen Proteins wurden im Hinblick auf katalytische Aktivität und zelluläre Lokalisation untersucht. Dazu wurden die ASM-Varianten in Expressionsvektoren kloniert und in einem Zellkulturmodell überexprimiert. Zwei der Varianten – p.P325A und p.G506R – zeigten im Vergleich zum ASM Wildtyp eine signifikant erniedrigte Enzymaktivität, während die Aktivität von p.A487V nicht verändert war. Alle drei untersuchten Varianten kolokalisierten mit LAMP1 in Lysosomen, so dass kein Unterschied im Vergleich zum Wildtyp beobachtet wurde. Im Rahmen der Arbeit wurden drei bislang nicht beschriebene ASM-Spleißvarianten identifiziert und kloniert. Die Spleißvarianten führten nach Überexpression nicht zu einem Anstieg der zellulären ASM-Aktivität. Es wurde allerdings beobachtet, dass sich der relative Ceramid-Gehalt der Zellen signifikant verringerte, was für eine dominant-negative Wirkung der Spleißvarianten spricht. Für die Spleißvariante ASM-5, die nur eine unvollständige katalytische Domäne aufweist, konnte gezeigt werden, dass es unter Serum-Entzug zu einer Translokation des Proteins ins Lysosom und zu einer Hemmung der zellulären ASM Aktivität kommt. Unter basalen Bedingungen hingegen lokalisierte das Protein im endoplasmatischen Retikulum und hatte keinen Einfluss auf die ASM-Aktivität. In RT-PCR Untersuchungen konnten die neuen Spleißvarianten bei gesunden Probanden nachgewiesen und eine Veränderung der Expression bei depressiven Patienten während der Therapie gezeigt werden. Die vorliegende Arbeit liefert erste Hinweise darauf, wie verbreitete ASM- Sequenzvariationen die ASM-Aktivität beeinflussen können. Weitere Arbeiten sollten zeigen, ob diese Mechanismen zur Entstehung der Majoren Depression beitragen.
12 Summary
Summary
In a pilot study conducted in 2005 by Kornhuber et al. , we detected increased activity of acid sphingomyelinase (ASM) in patients suffering from major depressive disorder compared to controls. In this study, we investigated the potential influence of ASM sequence variations on ASM activity to get insight into the causative mechanism of this association on the molecular level. On the one hand, we characterized the impact of generally occurring DNA sequence variations, which were also detected in depressed patients. On the other hand, we investigated ‘RNA sequence variations’, i.e. the role of alternative splicing for cellular ASM activity levels. Despite just slightly altered protein sequences, the resulting ASM variants could be prone to pathologic functioning. At the genomic level, we investigated the impact of three missense variations located in the coding region of the mature protein on enzymatic activity and cellular localization. Therefore, different ASM variations were cloned and over-expressed in a cell culture model. Compared to ASM wild type, two ASM variants – p.P325A and p.G506R – displayed significantly decreased ASM activity, whereas variation p.A487V did not change enzymatic activity. Localization patterns were unchanged, since all variants revealed co-localization with LAMP1 in lysosomes. In this study, three to date not described ASM splice variants were identified and cloned. Compared to ASM wild type, new splice variants did not increase cellular ASM activity levels upon over-expression. However, we detected a significant decrease in relative cellular ceramide levels, indicating a dominant-negative effect exerted by new ASM splice variants. For isoform ASM-5, which displays a disrupted catalytic domain, we could demonstrate translocation into lysosomes and the inhibition of cellular ASM activity upon serum starvation. In contrast, ASM-5 was located inside the endoplasmic reticulum and did not impact cellular ASM activity under basal conditions. By RT-PCR differential expression patterns of ASM splice variants could be detected in blood cells of healthy volunteers. In patients suffering from major depression expression patterns differed before and after therapy with antidepressant drugs. This study offers first evidence how common ASM sequence variations can impact ASM activity. Further studies should investigate if these mechanisms could account for the pathogenesis of major depression.
13 2. Introduction
2. Introduction
2.1 Lysosomal sphingolipid disorders of the brain
Groups of diseases, which we today call ‘lysosomal disorders’, had been clinically recognized around the turn of the nineteenth to the twentieth century (Tay, 1881; Sachs, 1887). To get insight into the molecular basis of these diseases, it still took to the 1960s (Hers, 1963) – upon the discovery of the lysosome (De Duve et al. , 1955). Lysosomal diseases are defined as a group of inherited metabolic diseases, which are characterized by the accumulation of non-metabolized macromolecules like lipids, glycoproteins or oligosaccharides inside the lysosomes. Notably, most of lysosomal disorders show pathology in the brain, leading to severe clinical symptoms (Meikle et al. , 1999). According to the stored material, a classification system was established, with lipid storage material and especially the class of sphingolipids being the most complex group. The so called ‘sphingolipidoses’ cover glycosphingolipid storage diseases and not further specified diseases, including deficiencies of acid sphingomyelinase (ASM) and acid ceramidase, which lead to Niemann-Pick (NPD) and Farber disease, respectively (Platt and Walkley, 2004, p.35). After the biochemical identification of the storage material, also the genetic background of these diseases could be identified. Most of the to date described 41 disorders are autosomal recessive diseases (Meikle et al. , 1999). Interestingly, not only lysosomal hydrolases are affected, but also a variety of proteins exerting influence on their cellular processing, e.g. endoplasmic reticulum (ER)- and Golgi-associated processing enzymes, protective proteins or transmembrane proteins (Walkley, 2001). Due to the discovery of the molecular mechanisms of the lysosomal disorders, a classification system on the basis of molecular defects evolved, which harboured chances for better understanding and potential treatment of lysosomal disorders (Platt and Walkley, 2004, p.41).
14 2. Introduction
2.1.1 Sphingolipids play diverse roles in cellular processes
The term ‘sphingolipids’ is derived from the enigmatic and therefore sphinx-like properties of complex lipids, which were firstly described in the 1870s after their discovery within brain extracts. Sphingolipids play diverse roles in cellular processes: On one hand as structural membrane components, on the other hand as signalling molecules that mediate cellular stress responses (Zeidan and Hannun, 2007b).
2.1.1.1 Sphingolipids: Functional diversity by structural diversity
Sphingolipids are a large class of polar and amphipatic lipids, which show structural diversity. Their common feature is the core component sphingosine, which results from the condensation of serine with fatty acid-Coenzyme A conjugates (Loeffler, 2003). An N-linked fatty acid which can be quite heterogenous gives rise to ceramide, which is the source for more complex sphingolipids: The head group can be derivatized with different moieties, whereas condensed glycans result in glycosphingolipids, condensed phosphorylcholine in sphingomyelin. The chemical core structure in combination with the variety of head groups makes sphingolipids a unique class of lipids with essential functions for cellular processes (Breslow and Weissman, 2010). Sphingolipids significantly affect both the structural and functional properties of cell membranes. For example, sphingomyelin is an important component of the outer cell membrane (Emmelot and Van Hoeven, 1975). In addition, many sphingolipids are bioactive signalling molecules which impact diverse cellular functions. As an example, ceramide and sphingosine-1-phosphate exert signalling properties in intracellular and extracellular context, respectively. Therefore, nowadays sphingolipids are not seen as merely structural and ‘passive’ molecules anymore, but take an important position as bioactive molecules (Breslow and Weissman, 2010).
15 2. Introduction
2.1.1.2 Roles of sphingolipids for the CNS
Though in most cell types sphingolipids constitute the minor component of cell membranes besides glycerophospholipids and cholesterol, they resemble the vast majority in nervous cells. For example, glycosphingolipids and sphingomyelin are abundant components of membranes in the nervous system with the latter’s name being derived from the frequent prevalence within myelin sheaths (Zheng et al. , 2006). Besides the structural functions, sphingolipids also exert important cell-biological functions in the CNS. They are important for maintaining the functional integrity of neurons concerning survival, cell death, migration and differentiation, synaptic functioning and also neuron-glia interactions (Piccinini et al. , 2010). These essential roles in signal transduction are exerted via two different paths: Concerning structural influence, they modulate properties of membrane-associated receptors due to alterations of membrane composition. Concerning functional influence, they act as precursors of bioactive lipid mediators. These differential actions lead to a structural and functional modification of neuronal plasticity (Haughey, 2010; Swartz, 2008; Day and Kenworthy, 2009; Owen et al. , 2009; Stahelin, 2009). For example, synaptic activity both at pre- and post-synapses is highly influenced by sphingolipids. Vesicle trafficking, -fusion and transmitter release are regulated by the bioactive sphingolipids ceramide, sphingosin and sphingosin-1-phosphate (Kajimoto et al. , 2007). Also, synapse formation and the location of receptors are affected by the action of ceramide (Rohrbough et al. , 2004). In total, a dysfunction of sphingolipid metabolism can severely influence synaptic integrity and neuronal functioning.
2.1.1.3 The lipid messenger ceramide
The most extensively studied sphingolipid ceramide acts as a lipid messenger, exerting diverse effects on cellular processes, negatively on proliferation (Boucher et al. , 1995; Coroneos et al. , 1995) and differentiation (Sallusto et al. , 1996; Zhang et al. , 1997), blocking of cell cycle events (Dobrowsky et al. , 1993) and apoptosis (Obeid et al. , 1993). Due to the activity of acid sphingomyelinase, ceramide is generated by the breakdown of sphingomyelin. According to the ‘ceramide / sphingosine-1-phosphate rheostat’ concept increased ceramide levels promote apoptosis whereas an increase of the phosphorylated metabolite of ceramide, sphingosine-1-phosphate, counters this effect by inducing 16 2. Introduction proliferation, which regulates the balance between cell death and cell growth (Spiegel and Merrill, 1996; Spiegel et al. , 1998). The dynamic balance between these bioactive lipids is influenced by the activity of acid ceramidase, sphingosine kinase, sphingosine 1-phosphate phosphatase, sphingosine-acetyltransferase and ASM. Among these enzymes, ASM plays an important role, since the first bioactive molecule within the rheostat is generated upon its enzymatic activity (Fig. 2.1).
Fig. 2.1 The ‘ceramide / sphingosine-1-phosphate rheostat’ concept. The dynamic balance between cell death and growth is highly influenced by acid sphingomyelinase (ASM) and acid ceramidase (AC). Ceramide is generated by ASM-induced hydrolysis of sphingomyelin and exerts pro- apoptotic effects (red). The opposite effect is mediated by sphingosine-1-phopohate (blue) (modified from Kornhuber et al. (2010)).
Ceramide impacts the signal transduction of the cell via the modulation of the cell membrane and via its function as lipid messenger. Alterations affecting the cell membrane can induce dramatic changes concerning the biophysical properties of membrane microdomains, which are often built by sphingomyelin and cholesterol (Simons and Ikonen, 1997; Kolesnick et al. , 2000). Ceramide molecules interact spontaneously and fuse to large macrodomains (Holopainen et al. , 1998; Kolesnick et al. , 2000; Nurminen et al. , 2002). These macrodomains play an important role in signal transduction processes and exert a general effect on diverse signal cascades. For example, the generation of ceramide-rich platforms leads to the reorganization and an increased density of receptors, e.g. of the death receptor CD95 and accessory signalling molecules, which means an amplification of this signal (Grassmé et al. , 2001a; Grassmé et al. , 2001b; Grassmé et al. , 2003). The generation of ceramide is probably an important cellular reaction of the cell on stressful events (Quintans et al. , 1994; Herr et al. , 1997). Therefore, ceramide as lipid messenger 17 2. Introduction exerts direct influence on different kinases like ceramide-activated proteinkinase, proteinkinase C ζ und ceramide-activated proteinphosphatase, which in turn impact signalling molecules like c-Raf and NF κb (Zhang et al. , 1997). Thus, ceramide exerts the function of an effector molecule, regulating many important downstream targets. In total, ASM generating increased levels of ceramide by hydrolizing sphingomyelin plays a key role for cellular sphingolipid metabolism and impacts diverse cellular processes.
2.1.2 Molecular defects of lysosomal enzymes
The lysosome is the only cellular organelle with an acidic milieu of pH 5 and is covered by a membrane bilayer. Since the lysosome contains a variety of hydrolases, it was seen as an ‘organelle for garbage disposal’, carrying out the catabolism of internalized material (Alberts, 2002; Loeffler, 2003): After the uptake of extracellular material, for example by receptor- mediated endocytosis, internalized macromolecules enter the endosomal-lysosomal pathway. On their way to the lysosome these molecules are partly degraded until they get their final destination. Inside the lysosome, final breakdown by diverse hydrolases takes place, completed with the storage of undigestable material (Platt and Walkley, 2004, chapter 1). But nowadays, the perception of the lysosome takes a turn: Instead of being there for the mere breakdown of molecules, it is supposed to be a source for many important regulative processes. For example, the lysosomal form of ASM generates ceramide, which impacts important signal cascades (Kolesnick, 1991). Therefore, molecular defects of lysosomal enzymes can be deleterious for proper cellular functioning. For neurons, the effectiveness of these processes is even more important, since they have special requirements for endocytic recycling and have to function during their whole longevity (Slepnev and De Camilli, 2000; Platt and Walkley, 2004, chapter 1). Molecular defects of lysosomal enzymes can occur at diverse processing levels, via mutations in the gene itself, abnormal processing of the protein in the ER and in the Golgi, missing protective or activating proteins or altered transport proteins (Platt and Walkley, 2004, chapter 4).
18 2. Introduction
2.1.2.1 Primary defects
Mutations in genes coding for lysosomal enzymes are the most common causes of lysosomal storage diseases. Among sphingolipidoses, many of the glycosphingolipid storage diseases are caused by primary enzymatic defects, e.g. Fabry disease resulting from mutations within α-galactosidase. To give examples for the not further distinguished sphingolipidoses, Niemann-Pick disease type A and B is caused by mutations in the sphingomyelin phosphodiesterase gene (SMPD1) coding for ASM, or Farber disease, which harbours mutations in the gene coding for acid ceramidase. In most of these disorders, the catalytic activity of the respective enzyme is abolished. Therefore, the catalytic function of an essential enzyme comprising the endosomal-lysosomal system is lacking (Platt and Walkley, 2004, chapter 4). The extent of the derived deleterious effect depends on the normal load of the respective substrate presented to the lysosome and the residual activity of the enzyme. The worst pathologic effects of primary defects are located in those tissues, where a certain substrate is subject to extensive catabolism, e.g. gangliosides in neurons. Also, the respective threshold of storage is of importance (Conzelmann and Sandhoff, 1983). But even if the main problems of storage arise in tissues outside the brain, also in neuronal and glial cells there probably is a steady build up of storage material, leading to clinical manifestation at a later stage of the process. Since these problems may be worse for long living neurons, all lysosomal enzyme defects could be seen as causes for brain diseases. Lysosomal hydrolases pass many stations from their synthesis in the rough ER to their destination, the lysosomes. Many post- translational modifications take place, the removal of the N-terminal signal peptide, acquisition of the mannose-6-phosphate recognition marker and glycosylation- and processing steps to reach their final conformation. Any of these steps can be affected by mutations at a certain position in the gene giving rise to the enzyme (Platt and Walkley, 2004, chapter 4). Different types of mutations have been linked to lysosomal enzyme deficiencies, for example SNPs leading to missense, nonsense or splice-site mutations, deletions, insertions or chromosomal rearrangements. Usually null alleles evolve, which result in the expression of non-functional enzymes. In some cases, a residual activity of the resulting enzymes is left. But a clear prediction of the effects of DNA variation on protein properties remains difficult (Terp et al. , 2002). As an exception, substitution of an amino acid which is directly involved in catalytic action or interaction with activator proteins probably leads to a lack of catalytic
19 2. Introduction activity. An important tool for detecting effects of DNA variation is the cloning of gene variants coding for altered proteins. In vitro expression analyses allow insights into the effects on enzymatic activity caused by mutations. However, if a missense mutation does not result in the expression of a catalytic inactive enzyme, the processing steps should be controlled before negating pathologic effects. Also, mutations are able to enhance or lessen the effects of another sequence variation (Islam et al. , 1996). Additionally, the clinical phenotype can give a hint for null alleles caused by mutations. Severely affected patients being homozygous for a mutation probably produce null allele enzymes, whereas heterozygous patients should be less affected then. Consequences which result from certain mutations, especially how they affect the structure, the intracellular transport and the activity of the resulting enzyme, are of high importance for enzyme properties and the following treatment of pathologic effects (Platt and Walkley, 2004, chapter 4).
2.1.2.2 Posttranslational defects
The lysosomal system can be disturbed also on the posttranslational level. For example, lysosomal processing of the enzymes in the ER can be involved. This is seen in the so far unique known molecular mechanism of a lysosomal storage disease, the multiple sulphatase deficiency (Hopwood and Ballabio, 2001). In this defect, the activating step of the posttranslational generation of a C α-formylglycine residue in the catalytic centre of sulfatases is lacking (von Figura et al. , 1998).
2.1.2.3 Defective trafficking
Defective trafficking can lead to lysosomal diseases, if the trafficking from ER to the lysosomal system via the Golgi apparatus is perturbed. For example, the I-cell disease, also called mucolipidosis type II, is characterized by a defective trafficking due to the lack of mannose-6-phosphate phosphorylation. Affected is the protein responsible for synthesis of the mannose-6-phosphate-recognition marker, the N-acetylglucosaminyl-1-phospho- transferase. Instead of trafficking to lysosomes, the enzymes are secreted (Kornfeld and Sly, 2001). Alternative routes for lysosomal targeting in addition to the mannose-6-phosphate receptor system are barely understood. Taken together, defects affecting the trafficking of 20 2. Introduction lysosomal enzymes can cause two different effects: The lysosome is deprived of an important hydrolase, and a lysosomal enzyme precursor is secreted, potentially exerting effects outside the cell (Platt and Walkley, 2004, chapter 6).
2.2 Acid sphingomyelinase in health and disease
Acid sphingomyelinase (EC 3.1.4.12) is a glycoprotein located mainly in the lysosome and catalyses the breakdown of sphingomyelin into ceramide and phosphorylcholine at a pH optimum of pH 5 (Quintern et al., 1987). As one important function, ASM activity changes the lipid composition of membranes. A second important function is the generation of the bioactive lipid ceramide. ASM exerts biomedical significance, since both increase and decrease in ASM activity leads to severe pathologic effects. Several sequence variations of ASM cause the loss of enzymatic activity, resulting in Niemann-Pick disease type A and B. Since ASM plays a strategic role in cellular stress response, its tight regulation is crucial for cellular functioning.
2.2.1 Acid Sphingomyelinase – the lysosomal enzyme for sphingomyelin breakdown
ASM was firstly described in 1966 by Kanfer et al. (1966), who reported a sphingomyelin- cleaving enzyme in rat liver tissue. The subcellular localization of ASM inside the lysosome was identified two years later (Weinreb et al. , 1968). But it still took until 1991 to clone ASM (Schuchman et al. , 1991b), which was the starting point to investigate biochemical properties of the enzyme and the molecular mechanisms of ASM regulation.
21 2. Introduction
2.2.1.1 Genomic organization and transcription
The gene coding for ASM, SMPD1 , is located on chromosome 11p15.1-p15.4, covering about 5000 base pairs (da Veiga Pereira, 1991). Interestingly, it gives rise to two different enzymes, the lysosomal and the secreted sphingomyelinase, which probably play different metabolic roles (Schissel et al. , 1996). Transcription of ASM is regulated by different upstream promotor elements, including a TATA-box and binding sites for transcription factors SP1 and AP-2 (Langmann et al. , 1999). The cDNA synthesized from human mRNA displays an untranslated region of 174 and 388 base pairs at the 5’ and the 3’ end, respectively. The SMPD1 open reading frame covers 1896 base pairs coding for 631 amino acids. On the posttranscriptional level, ASM is subject to alternative splicing events. For the lysosomal form of ASM, four different transcripts are identified so far (Quintern et al. , 1989; Schuchman et al. , 1991b; Schuchman et al. , 1992). The ‘regularly’ spliced transcript, termed ASM type 1 (GenBank Accession Number NM_000543.4; here referred to as ASM-1) is composed of exons 1 to 6, with the corresponding five introns spliced out. Compared to this reference, alternatively-spliced transcript ASM type 2 (GenBank Accession Number NM_001007593.1; here ASM-2) displays a specific insertion of 40 bp derived from intron 2 and lacks exon 3. Alternatively-spliced transcript ASM type 3 (GenBank Accession Number NR_027400; here ASM-3) is lacking the complete exon 3 which creates a frameshift and subsequently leads to a premature stop codon. An additional ASM transcript, which lacks 652 bp of exon 2, was isolated from human brain tissue (GenBank Accession Number AY649987.1; here typed ASM-4) (Fig. 2.2). According to Schuchman and colleagues (Quintern et al. , 1989; Schuchman et al. , 1991b), ASM-1 occurs at a frequency of about 90%, ASM-2 and -3 of about 10%.
22 2. Introduction
Fig. 2.2 ASM is subject to alternative splicing events. Model of genomic alignment. Compared to ASM-1, ASM-2 displays a specific insertion of 40 bp and lacks exon 3. ASM-3 is lacking exon 3, whereas ASM-4 lacks 652 bp of exon 2, both creating a frameshift and a premature stop codon. In the depicted models of ASM exon-intron structure, the line represents the genomic sequence while black boxes stand for exons translated in ASM-1, grey boxes for exonic sequence translated into different amino acids due to a frameshift or sequence corresponding to introns in ASM-1, striped boxes for lacking exonic sequence and white boxes for exonic sequence following a premature termination codon (indicated by asterisk). Scheme is drawn to scale.
2.2.1.2 Sequence variations of Acid Sphingomyelinase
According to GenBank database SNP, 32 SNPs within the ASM coding sequence are identified. The most frequent SNPs are c.107T>C / p.V36A with a frequency of 0.32 and c.1522G>A / p.G506R with a frequency of 0.251. Besides the different SNPs there also exists a polymorphic part located in the region coding for the signal peptide of ASM, which is characterized by a three- to eight-fold repeat of the hexanucleotide GCTGGC (c.108GCTGGC[6] / p.37(LA)3-8) (Wan and Schuchman, 1995).
2.2.1.3 Properties of the Acid Sphingomyelinase protein
The ASM protein displays several domains. The enzyme is composed of the putative signal peptide (aa 1-48), a saposin-B-domain for proper substrate interaction (aa 91-167), a proline- rich domain (aa 168-200), a calcineurine-like phosphoesterase domain considered as the catalytic centre (aa 201-463) and a carboxy-terminal domain (aa 464-631) (Marchler-Bauer A, 2007; Jenkins et al. , 2009) (Fig. 2.3).
23 2. Introduction
Fig. 2.3 ASM is composed of several domains. Schematic model of ASM protein structure. Shown are different domains with respect to amino acid numbers. Mannose-6-phosphate sites are indicated by symbols. Scheme is drawn to scale.
ASM is subject to several steps of processing before the mature enzyme is obtained. In a first step, ASM is translated into a premature pre-pro-polypeptide with an apparent molecular mass of 75 kDa (64 kDa in deglycosylated state), which is found in the ER and does not exert catalytic activity (Ferlinz et al. , 1994; Hurwitz et al. , 1994b). As usual for proteins located within subcellular organelles, also ASM displays an N-terminal signal peptide. In comparison to ‘regular’ signal peptides of about 20 amino acids, the ASM signal peptide seems to be special due to its length of 48 amino acids (Garnier et al. , 1978; Schuchman et al. , 1991b; Wan and Schuchman, 1995). According to the polymorphism on the genomic level, the motive composed of leucine and alanine (‘LA’) occurs to a different extent, being repeated three, four, five, six or eight times (Wan and Schuchman, 1995) and probably influences the hydrophobicity of the ASM N-terminal region. The signal peptide plays an important role for the functioning of ASM. Is the signal peptide lacking, ASM does not reach the lysosomal compartment: Neither is it synthesized into the lumen of the ER, nor is it modified properly and trafficked by vesicular transport. Instead, ASM is localized inside the cytoplasm (Ferlinz et al. , 1994). Besides proper localization, the signal peptide is also essential for generating catalytic active ASM enzymes. Recombinant ASM constructs without the sequence coding for the signal peptide are catalytically inactive in vitro , probably due to the lack of glycosylation and zinc acquisition. Also, no ASM activity can be detected if sorting and trafficking is altered (Ferlinz et al. , 1994; Ni, 2006). In total, ASM targeting to lysosomes is essential for proper enzymatic functioning, which stresses the importance of the ASM signal peptide. Therefore, the signal peptide can influence ASM activity, even though it is not present in the mature ASM protein. Within the ER and Golgi further processing of ASM takes place. After cleavage of the signal peptide inside the ER, a 72 kDa precursor form of ASM occurs (61 kDa in deglycosylated
24 2. Introduction state) – yet without being catalytically active. This precursor protein gives rise to two distinctly processed ASM forms. The major part is processed to a 70 kDa form and associated with a zinc ion. This mature ASM protein is localized inside the lysosome and exerts catalytic activity. In a further processing step inside the lysosome, the 70 kDa form is cleaved to a 52 kDa form. The minor part of the 72 kDa precursor is cleaved already inside the ER and Golgi to a 57 kDa form, which is not glycosylated and rapidly degraded (Ferlinz et al. , 1994). In total, processing of lysosomal ASM is essential for generating the properly localized and catalytically active enzyme. In contrast, secreted sphingomyelinase is not further processed and is detected as 75 and 57 kDa forms when secreted into extracellular space.
2.2.2 Regulation of Acid Sphingomyelinase activity
ASM plays a role for diverse physiological processes. A variety of external stimuli cause an activation of ASM, e. g. CD95 ligand (Lin et al. , 2000), lipopolysaccharide (Haimovitz- Friedman et al. , 1997), ionizing radiation (Paris et al. , 2001), UV-radiation (Charruyer et al. , 2005), cisplatin (Rebillard et al. , 2008) or tumor necrosis factor-α (Garcia-Ruiz et al. , 2003) to name a few.
2.2.2.1 Transcriptional level
Different settings lead to an up-regulation of ASM at the transcriptional level (Lecka-Czernik et al. , 1996; Langmann et al. , 1999; Murate et al. , 2002; Wu et al. , 2005; Shah et al. , 2008), representing a slow regulative response to external stress stimuli. For example, differentiation of macrophages is correlated with an up-regulated expression of ASM, since transcription factors AP-2 and Sp1 are essential for both mechanisms (Langmann et al. , 1999).
25 2. Introduction
2.2.2.2 Posttranscriptional level
Since four different ASM transcripts have been identified to date (see 2.2.1.1), a further aspect of ASM regulation could be seen in alternative splicing. Thus far, only the full-length variant ASM-1 has been proven to encode for an active enzyme, whereas ASM-2 and ASM-3 were catalytically inactive, since both encoded proteins show a disrupted catalytic domain and the latter one additionally lacks the carboxy-terminal domain due to truncation (Schuchman et al. , 1991b). ASM-4 is lacking parts of the saposin-B-, the catalytic and the carboxy-terminal domain, but its biochemical properties have not been determined so far. Since only a single ASM splice variant is functional, cellular regulation of ASM at the RNA level is highly assumed (Zeidan and Hannun, 2010). Mechanisms underlying alternative splicing of ASM remain unknown and have to be elucidated.
2.2.2.3 Posttranslational modifications
In contrast to the slow transcriptional response to external stress stimuli, posttranslational modifications serve as a fast regulative response of ASM. Activation of ASM in vitro results from interaction with zinc (Schissel et al. , 1998) and copper ions, the latter causing dimerisation of two ASM molecules via the most carboxy-terminal located cysteine at position 631 (Qiu et al. , 2003). Also, an activating effect is exerted by protein kinase δ, which phosphorylates the serine residue at position p.508 in the carboxy-terminal domain of ASM (Zeidan and Hannun, 2007a).
2.2.2.4 Regulation via degradation
Degradation of ASM is mediated by antidepressant drugs. Different classes of antidepressant drugs inhibit ASM activity (Kornhuber et al. , 2008). These weak organic bases like desipramine and imipramine act as functional inhibitors of ASM, since no direct inhibition is exerted (Sakuragawa et al. , 1977; Yoshida et al. , 1985; Albouz et al. , 1986; Kornhuber et al. , 2005). Rather, these antidepressants are supposed to impact the attachment of ASM to the intra-lysosomal membrane. Thus, ASM is detached from the membrane and probably
26 2. Introduction subjected to proteolytic cleavage inside the lysosome (Hurwitz et al. , 1994a; Kölzer et al. , 2004). Therefore, it is supposed that ASM activity is regulated via proteolytic degradation. Evidence was offered by experiments using protease inhibitors, which were able to abolish effects exerted by antidepressants (Hurwitz et al. , 1994a).
2.2.3 Acid Sphingomyelinase and pathologic context
Imbalanced ASM activity is involved in different pathologic contexts. Either way of ASM dysregulation induces pathologic effects – decreased as well as increased ASM activity.
2.2.3.1 Decreased activity of Acid Sphingomyelinase
Decreased ASM activity is the cause of Niemann-Pick disease type A (OMIM: 257200) and B (OMIM: 607616), which is an autosomal recessive sphingolipidosis. Deficient ASM activity occurs due to inherited sequence variations in the SMPD1 gene coding for ASM (Brady et al. , 1966). Type A is a severe infantile neurodegenerative disease, leading to death within three years of life. It is characterized by massive hepatosplenomegaly and psychomotor deterioration. In contrast, type B does not display neurological involvement, but is a visceral form featuring hepatosplenomegaly and affecting the reticuloendothelial and respiratory system. Patients survive into adulthood. As detected within cultured fibroblasts, the ASM activity of patients is about 1-5% of normal activity levels (Levran et al. , 1991). On the genetic level, a variety of ASM sequence variations causing NPD type A and B are known to date. Different genotypes lead to missense mutations, for example p.M381I, p.R496L, p.G577S and p.L302P, which cause the loss of enzymatic activity, resulting in NPD type A. Missense mutations p.R608del, p.S436R, p.G242R, p.N383S retain at least partial catalytic activity and cause NPD type B, to name a few. Notably, mutations affect especially the integrity of the catalytic and the C-terminal domain (Ferlinz et al. , 1991; Levran et al. , 1991; Levran et al. , 1992; Takahashi et al. , 1992a; Takahashi et al. , 1992b). In total, genetic variation of ASM causing NPD makes clear that SNPs located within the SMPD1 gene can severely affect ASM activity.
27 2. Introduction
2.2.3.2 Increased activity of Acid Sphingomyelinase
An increase in ASM activity – the opposite effect as seen in NPD – is associated with different diseases. A common feature of these diseases is the involvement of cellular stress, which in turn leads to elevated ceramide levels via the activation of ASM. Ceramide is supposed to be the cause for apoptosis of hepatocytes as described for Morbus Wilson (Lang et al. , 2007) and for the onset of lung odems occurring during acute lung injuries (Goggel et al. , 2004). Interestingly, ASM knock out mice survive much longer after LPS induced endotoxic shock syndrome compared to wild type mice (Haimovitz-Friedman et al. , 1997). Other diseases displaying elevated ASM activity levels are cystic fibrosis (Teichgräber et al. , 2008) and arteriosclerosis (Tabas, 2004). Also patients suffering from different neuro- psychiatric disorders like Alzheimer’s dementia (He et al. , 2008), status epilepticus (Mikati et al. , 2008) and major depressive disorder (Kornhuber et al. , 2005) show increased ASM activity levels. The underlying mechanisms causing increased ASM activity in so many different pathologic contexts remain unclear. But considering the correlation between unbalanced ASM activity and pathologic consequences, a tight regulation of ASM is essential for intact cellular functioning.
2.3 The role of Acid Sphingomyelinase in Major Depressive Disorder
Major depressive disorder (MDD) is a severe psychiatric disorder characterized by pathologic changes concerning mood and motivation. According to ICD-10, following symptoms serve for the diagnosis of MDD: Episodes of sadness, loss of interest, pessimism, negative beliefs about the self, decreased motivation, behavioural passivity, changes in sleep, appetite and sexual interest, and suicidal thoughts and impulses (DeRubeis et al. , 2008). Patients suffering from MDD show divergent patterns of symptoms, varying from somatic, psychomotor, emotional, motivational, cognitive to social components (Davison, 1998; Hautzinger, 2003). Prevalence for the onset of MDD is about 5% with women being affected much more frequently than men (8% versus 3%) at averaged age between thirty and forty (Davison, 1998).
28 2. Introduction
The etiopathology of MDD is not clear to date. Several correlations of MDD with biological changes within patients are identified, but do not serve as causative factors. The current etiopathic model is the diathesis-stress-model: On the genetic level, certain dispositions are inherited, leading to a potential vulnerability. Environmental factors like stress in combination with this vulnerability trigger the manifestation of disease pattern. In depression, altered genetic disposition concerns the neurotransmitter systems, certain hormonal systems and circadian rhythms, which is affirmed by experimental data on the morphological, functional and biochemical level (Köhler, 2005).
2.3.1 Results of the pilot study
In our pilot study, Kornhuber and colleagues compared the level of ASM activity in peripheral mononuclear cells (PBMCs) between patients suffering from MDD (n=17) and healthy volunteers (n=8). It was shown that depressed patients displayed a significantly higher level of ASM activity compared to controls. Also, the severity of depression, indicated by the Hamilton Scale of Depression (17-HDRS), and ASM activity levels were positively correlated (Kornhuber et al. , 2005). The relation between major depression and ASM activity is strengthened by findings concerning antidepressant drugs, since different antidepressant drugs like desipramine and imipramine inhibit ASM activity (Albouz et al. , 1986). This effect was also observed during our pilot study. Cultured PBMCs from healthy volunteers were treated with imipramine or amitriptyline, which resulted in a clear decrease of ASM activity. After removal of drugs, ASM activity raised to normal levels again (Kornhuber et al. , 2005). According to our pilot study, there is a clear link between the psychiatric disease MDD and elevated ASM activity levels. The type of interrelation however remains to be elucidated.
29 2. Introduction
2.3.2 Sequence variations of Acid Sphingomyelinase in depressed patients
Due to the results of the pilot study, we started to investigate the molecular mechanisms of increased ASM activity levels in depressed patients. A possible reason for elevated ASM activity levels could be seen in the enzyme itself. Analogous to the primary defect of ASM causing NPD, also sequence variations leading to an increased intrinsic activity could exist, as was reported for other enzymes, e. g. thermolysin (Kusano et al. , 2010) or tripeptidyl peptidase I (Walus et al. , 2010). DNA sequence variations could lead to ASM proteins which are prone to dysregulated activation and might predispose to psychiatric and neurological diseases. Therefore, we investigated a collective of 58 patients suffering from major depression for ASM sequence variations. We mapped the genetic variation of SMPD1 by sequencing the complete gene locus including the adjacent 5’ area. Several variations were discovered: 15 common SNPs with a minor allele frequency of >0.1; seven rare SNPs with a minor allele frequency of <0.05; a common 5-bp deletion and a common 6-bp repeat; a GC-rich region preceding exon 1 and two poly-T stretches in intron 2. Among these variations were three missense variations leading to an altered mature protein sequence. To get insight into the potential effect of those DNA sequence variations on the properties of ASM enzymes, it would be inevitable to investigate ASM variants with respect to their functionality. Due to amino acid exchange, different properties of the enzyme could be altered, for example enzymatic activity and protein trafficking.
2.4 Aims of the study
As detected in the pilot study, depressed patients display enhanced activity of ASM within their blood cells. To gain insight into the causative mechanism of elevated ASM activity levels, different ASM sequence variations should be investigated. Despite the just slight differences, a change of protein sequence could make these ASM proteins prone to pathologic functioning. ASM variants based on three different DNA sequence variations detected within depressed patients should be characterized concerning their catalytic activity and cellular localization. According to their alterations in protein sequence, differential characteristics of these variants were supposed.
30 2. Introduction
To test if alternative splicing is involved in the regulation of ASM activity, also ASM isoforms resulting from alternative splicing events should be characterized. Besides their catalytic activity and cellular localization, expression patterns should be investigated. It was supposed that alternatively spliced ASM variants could contribute to the regulation of cellular ASM activity levels. For these purposes, different ASM variants should be cloned and expressed as recombinant fusion proteins. Using a cell culture model, their catalytic activity should be assayed and their cellular localization determined. For expression analyses a RT-PCR system should be established. The aim of the study was to get a specific characteristic for each of the different ASM variants. This would mean a deeper insight into the mechanism of dysregulated ASM activity.
31 3. Material and Methods
3. Material and Methods
3.1 Material
Technical equipment, auxiliary material and chemicals can be found in the appendix.
3.1.1 Enzymes
Restriction enzymes NEB, Frankfurt, Germany Shrimp alkaline phosphatase Roche, Mannheim, Germany T4-DNA-ligase Invitrogen, Darmstadt, Germany KAPA HiFi DNA polymerase Peqlab, Erlangen, Germany Tfi DNA polymerase Invitrogen, Darmstadt, Germany PfuUltra II Fusion HS DNA Polymerase Stratagene, La Jolla, CA, USA In-Fusion Advantage PCR Cloning Kit Clontech, Mountain View, CA, USA VILO cDNA reaction kit Invitrogen, Darmstadt, Germany
3.1.2 Antibodies
3.1.2.1 Primary antibodies
Tab. 3.1 Primary antibodies in cotext of their application. WB means application within Western Blot analyses, ICC within immunocytochemistry analyses. Antibody Species Application Source Diluted in FLAG M2 mouse WB Sigma 0.5 % Milk /PBS/T 1:1000 F1804 GAPDH mouse WB Millpore 0.5 % Milk /PBS/T 1:200.000 MAB1374 LAMP1 mouse ICC DSHB Iowa 0.5 % fish skin 1:100 H4A3 gelatine buffer / PBS PDIA4 rabbit ICC Proteintech 5% normal goat 1:100 14712-1-AP serum /PBS
32 3. Material and Methods
3.1.2.2 Secondary antibodies
Tab. 3.2 Secondary antibodies in cotext of their application. WB means application within Western Blot analyses, ICC within Immunocytochemistry analyses. Antibody Species Application Source Diluted in anti-mouse IgG goat WB Calbiochem 0.5 % Milk /PBS/T 1:20.000 401253 FITC anti-rabbit goat ICC Calbiochem 0.5 % Milk /PBS/T IgG 1:100 401214
3.1.3 Vectors and plasmids
3.1.3.1 Vectors
Vectors used in this study are shown in tab. 3.3.
Tab. 3.3 Vectors used in this study. Vector Size Resistance Reference pSC-B 3466 bp Ampicillin Stratagene, La Jolla, USA Flag-N2 4016 bp Kanamycin M. Reichel, Erlangen pmCherry-N1 4699 bp Kanamycin Clontech, Mountain View, USA
3.1.3.2 Plasmids
Plasmids used for expression studies are shown in tab. 3.4.
33 3. Material and Methods
Tab. 3.4 Plasmids used in this study. Name Insert Size Reference Description pASM_1522A_FLAGN2 ASM G506R 5.9 kb this study ASM wild type C-terminal FLAG epitope pASM_1522G_FLAGN2 ASM wild type 5.9 kb this study ASM G506R C-terminal FLAG epitope pASM_1522A_M381I_FLAGN2 ASM 5.9 kb this study ASM G506R/M381I C-terminal FLAG G506R/M381I epitope pASM_1522G_M381I_FLAGN2 ASM M381I 5.9 kb this study ASM M381I C-terminal FLAG epitope pASM_1522A_S508A_FLAGN2 ASM 5.9 kb this study ASM S508A/G506R C-terminal FLAG S508A/G506R epitope pASM_1522G_S508A_FLAGN2 ASM S508A 5.9 kb this study ASM S508A C-terminal FLAG epitope pASM_1522A_P325A_FLAGN2 ASM 5.9 kb this study ASM P325A/G506R C-terminal FLAG P325A/G506R epitope pASM_1522G_P325A_FLAGN2 ASM P325A 5.9 kb this study ASM P325A C-terminal FLAG epitope pASM_1522A_A487V_FLAGN2 ASM 5.9 kb this study ASM A487V/G506R C-terminal FLAG A487V/G506R epitope pASM_1522G_A487V_FLAGN2 ASM A487V 5.9 kb this study ASM A487V C-terminal FLAG epitope pASM_1522A_splvarIV_FLAGN2 ASM-5 5.8 kb this study ASM-5 C-terminal FLAG epitope
ASM_1522A_splvarV_FLAGN2 ASM-6 5.5 kb this study ASM-6 C-terminal FLAG epitope pASM_1522A_splvarVI_FLAGN2 ASM-7 5.2 kb this study ASM-7 C-terminal FLAG epitope pASM_1522A_cherryN1 ASM G506R 6.6 kb this study ASM G506R C-terminal cherry epitope pASM_1522G_cherryN1 ASM wild type 6.6 kb this study ASM wild type C-terminal cherry epitope pASM_1522A_M381I_cherryN1 ASM 6.6 kb this study ASM G506R/M381I C-terminal cherry G506R/M381I epitope pASM_1522G_M381I_cherryN1 ASM M381I 6.6 kb this study ASM M381I C-terminal cherry epitope pASM_1522A_S508A_cherryN1 ASM 6.6 kb this study ASM S508A/G506R C-terminal cherry S508A/G506R epitope pASM_1522G_S508A_cherryN1 ASM S508A 6.6 kb this study ASM S508A C-terminal cherry epitope pASM_1522G_P325A_cherryN1 ASM P325A 6.6 kb this study ASM P325A C-terminal cherry epitope pASM_1522G_A487V_cherryN1 ASM A487V 6.6 kb this study ASM A487V C-terminal cherry epitope pASM_1522A_splvarIV_cherryN1 ASM-5 6.5 kb this study ASM-5 C-terminal cherry epitope pASM_1522A_splvarV_cherryN1 ASM-6 6.2 kb this study ASM-6 C-terminal cherry epitope pASM_1522A_splvarVI_cherryN1 ASM-7 5.9 kb this study ASM-7 C-terminal cherry epitope
34 3. Material and Methods
3.1.4 Biological material
3.1.4.1 Bacteria
For transformation XL1-Blue bacteria from Stratagene (La Jolla, CA, USA) were used in this work.
3.1.4.2 Eucaryotic cells
The neuroglioma cell line H4, derived from brain of a caucasian male and purchased from Promochem (Wesel, Germany) and the adenocarcinoma cell line HeLa, derived from cervix of a black female and purchased from the American type culture collection (ATCC, Wesel, Germany) were used in this work.
3.1.5 Buffers and solutions
Reagent-grade water from a MilliQ deionizator (Millipore, Eschborn, Germany) with a specific resistance set to 18.2MW/cm 3 was used for buffers, solutions and dilutions.
BES-Buffer BES 2.14 g NaCl 3.28 g
Na 2HPO 4x2H 2O 0.054 g adjust to pH 6.98 using NaOH
ad 200 ml using H 20bidest
35 3. Material and Methods
0.2 % Fish skin gelatine buffer BSA Fraktion V 2.5 g Fish Skin Gelatine 0.5 g Triton X-100 0.25 g ad 250 ml using 1xPBS
Glycerol solution Glycerol 65 ml / 81.99 g
1 M MgSO 4 10 ml 1 M Tris / HCl pH 8.0 2.5 ml
Ad 100 ml using H 20bidest
10 x Laemmli buffer Tris 30 g Glycin 144 g SDS 10 g
ad 1000 ml using H 20bidest
LB medium Trypton 10 g Yeast extract 5 g NaCl 5 g adjust to pH 7.0 using HCl
ad 1000 ml using H 20bidest
4x Lower gel buffer (1.5 M Tris-HCl, pH 8.8) Tris 18.15 g
H20bidest 50 ml adjust to pH 8.8 using 6 N HCl
ad 100 ml using H 20bidest
36 3. Material and Methods
Lysis buffer 1 M NaAc pH 5.0 2.5 ml 0.5 M EDTA 26 µl 2% NP40 500 µl 1 Tab. Complete w/o EDTA
ad 10 ml using H 20bidest
PBS / T 10x PBS 500 ml 100x Tween 2050 ml
ad 5000 ml using H 20bidest
RIPA buffer 1.5 M Tris / HCl (pH7.5) 3333 µl 5 M NaCl 3003 µl NP40 (Igepal CA-630) 1 ml 10% Na-Desoxycholat 5 ml 1% SDS 10 ml 10 Tab. Complete w/o EDTA
ad 100 ml using H 20bidest
4 x SDS running buffer Upper gel buffer (0.5 M Tris-HCl, pH 6.8) 5.0 ml Glycerol 5.04 g SDS 0.8 g DTT (MW 154.3 g/mol) 0.62 g Bromphenol blue 200 µl
ad 10 ml using H 20bidest
37 3. Material and Methods
SOC medium Yeast extract 0.5 g Trypton 2 g
MgCl 2 0.2 g
MgSO 4 0.25 g Glucose 0.36 g
ad 100 ml using H 2Obidest
10 x TBS Tris 60.55 g NaCl 87.66 g
ad 1000 ml using H 20bidest adjust to pH 7.5 - 7.6 using HCl
20 x TBE Tris 432 g Boric acid 220 g 0.5 M EDTA pH 8.0 160 ml
ad 2000 ml using H 20bidest
10 x Towbin-Puffer Tris 30 g Glycin 144 g adjust to pH 8.6
ad 1000 ml using H 20bidest
1 x Towbin-Puffer / 20% MetOH 10x Towbin Puffer 100 ml Methanol 200 ml
ad 700 ml using H 20bidest
38 3. Material and Methods
4x Upper gel buffer (0.5 M Tris-HCl, pH 6.8) Tris 6.05 g
H20bidest 60 ml adjust to auf pH 6.8 using 6 N HCl
ad 100 ml using H 20bidest
2 x YT-Medium Trypton 64 g Yeast extract 40 g NaCl 20 g adjust to pH 7.0 using 5 N NaOH
ad 4000 ml using H 20bidest
3.2 Methods
3.2.1 Cell-biological methods
3.2.1.1 Cell culture
Human neuroglioma cells (H4) were cultivated in DMEM medium supplemented with 10% (v/v) FCS and 4 mM L-glutamine. HeLa cells were cultivated in DMEM medium supplemented with 10% (v/v) FCS and 2 mM L-glutamine. Starving medium was lacking FCS and consisted of DMEM medium with 4 mM or 8 mM L-glutamine, respectively. Cells were kept at 37 °C in a humidified atmosphere at 8.5% CO 2 and were routinely split at a ratio of 1:4. Regular tests monitored by the MycoAlert Mycoplasma Detection Kit (Lonza, Cologne, Germany) for mycoplasma infection were always negative. All reagents used for cell culture were purchased from Biochrom (Berlin, Germany).
39 3. Material and Methods
3.2.1.2 Transient expression of ASM
For transfection, 1.5x10e 5 H4 cells / 9.6 cm 2 and 1x10e 5 HeLa cells / 9.6 cm 2 were seeded in 6-well plates with 2 ml culture medium. After 24 hours cells were grown to a confluence of about 60%. Cells were transfected using calcium phosphate precipitation. 7.5 µg plasmid were diluted in 90 µl H 2O and mixed with 10 µl of 2.5 M calcium chloride, then added to 100 µl of 2x N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer [50 mM BES,
280 mM NaCl, 1.5 mM Na 2HPO 4, pH 6.98; all obtained from Sigma-Aldrich (Munich, Germany)] during softly vortexing, incubated for 20 min at room temperature and added to the cells. The culture medium was changed 17 h after transfection including a washing step using TBS buffer. For experiments under serum-starving conditions 48 h upon transfection cells were shifted to serum-free medium for 24h. Transfection efficiency was about 60-70% as determined from the number of GFP-positive cells using the expression vector pmaxFP- green-C (Lonza, Cologne, Germany) in a parallel control transfection.
3.2.1.3 Immunocytochemistry
3x10e 4 H4 cells / 2 cm 2 were grown on glass slides in a 24-well dish (Nunc, Wiesbaden, Germany) with 0.5 ml culture medium. 48 h upon transfection with ASM constructs cells were washed with PBS (Gibco, Darmstadt, Germany) and fixed with 10% formalin for 15 min at room temperature. The slides were washed three times with PBS and permeabilized with 0.1% Triton-X in PBS for 10 min. For LAMP1 staining, cells were blocked with 0.2% Fish Skin Gelatine Buffer for 10 min and stained with anti-LAMP1 antibody (1:100; mouse) for 2 h at room temperature. For PDIA4 staining, cells were blocked with 5 % goat normal serum for 10 min and stained with anti-PDIA4 antibody (1:100; rabbit) for 2 h at room temperature. For FLAG staining, cells were blocked with 10 % FCS for 10 min at room temperature and stained with anti-FLAG antibody (1:500; mouse) for 3-5 h at room temperature. After three washes with PBS, cells were incubated with goat-anti-mouse (1:100) and goat-anti-rabbit (1:100) secondary antibodies coupled to FITC, respectively, for 2 h at room temperature in indicated solutions. After three final washes with distilled water slides were mounted in DAPI containing Fluorescent Mounting Medium (Dako, Hamburg, Germany) and analyzed by fluorescence microscopy (Axiovert 135, Zeiss, Jena, Germany) and laser scanning confocal microscopy (DM IRE2 / TCS SL, Leica, Solms, Germany).
40 3. Material and Methods
3.2.1.4 Preparation of whole cell extracts
48 h or 72 h after transfection cells were washed with PBS (Gibco, Darmstadt, Germany). For assaying ASM activity cells were lysed in 100 µl lysis buffer / 9.6 cm 2 [250 mM sodium acetate, pH 5.0 (Merck, Darmstadt, Germany), 0.1% NP-40 (Sigma-Aldrich, Munich, Germany), 1.3 mM EDTA (Sigma-Aldrich, Munich, Germany), 1 tablet of complete mini protease inhibitor mix/10 ml (Roche, Mannheim, Germany)]. For western blot analysis cells were lysed in 100 µl RIPA buffer / 9.6 cm 2 [50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, all obtained from Sigma-Aldrich (Munich, Germany), and complete protease inhibitor tablets from Roche (Mannheim, Germany)] for 20 min at 4 °C. Cell debris was removed by centrifugation at 10.000 g for 15 min at 4 °C. For MALDI-TOF MS analyses cells were washed in PBS (Gibco, Darmstadt, Germany) 24 h or 48 h after transfection, lysed in 100 l of MALDI- lysis buffer / 9.6 cm 2 [20 mM Tris/HCl pH 7.4, 100 M butylated hydroxytoluene (spectrometric grade, Sigma-Aldrich, Munich, Germany)], immediately frozen in liquid nitrogen and stored at -80 °C.
3.2.2 DNA technology
3.2.2.1 PCR for cloning approach
For cloning into pSC-B vector full-length ASM transcripts were amplified by PCR in a total reaction volume of 50 µl with 1 µl of undiluted cDNA, 1 U KAPA HiFi DNA polymerase with GC-buffer, 1.7 mM MgCl 2, 0.3 mM dNTPs (Peqlab, Erlangen, Germany) and 0.24 pmol/µl oligonucleotide primers (Operon, Ebersberg, Germany). For sub-cloning into FLAG- N2 expression vector 500 ng plasmid DNA were used as template. Primers flanking the ATG initiation codon and the stop codon according to the reference sequence NM_000543.4 carried Bsp E1 and Bgl II restriction sites (Tab. 3.5, indicated in bold). For truncated variants specific reverse primers were used (Tab. 3.5). Cycle parameters were as follows: initial
41 3. Material and Methods denaturation at 95 °C for 2 min, 35 cycles at 98 °C for 20 sec, 73 °C for 15 sec, 68 °C for 1 min, and a final extension at 68 °C for 5 min. PCR products were purified using the QIAquick PCR purification kit or QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to manufacturer’s recommendations.
Tab. 3.5 Oligonucleotides used for cloning of ASM into pSC-B vector and FLAG-N2 expression vector. ASM variant Primer Sequence 5’ -> 3’ ASM full length Forward TCCC TCCGGA ATGCCCCGCTACGGAGC variants Reverse GCG AGATCT GCAAAACAGTGGCCTTGG ASM-6 Forward TCCC TCCGGA ATGCCCCGCTACGGAGC Reverse GGA AGATCT ACGGGAACAAAAATTCATATGAAGAGAGAGG ASM-7 Forward TCCC TCCGGA ATGCCCCGCTACGGAGC Reverse GGA AGATCT CGGGAACAAAAATTCATATTGAGAGAGATG
3.2.2.2 Sub-cloning of ASM into pmCherry-N1 expression vector
Cloning ASM variants from FLAG-N2 expression vector into pmCherry-N1 expression vector for better tracking due to the fluorescent epitope was carried out using In-Fusion Advantage PCR Cloning Kit according to the manufacturer’s instructions (Clontech, Mountain View, CA, USA). Primers for this procedure are shown in table 3.6. Cycle parameters were as follows: initial denaturation at 95 °C for 2 min, 35 cycles at 98 °C for 20 sec, 68 °C for 15 sec, 68 °C for 1 min, and a final extension at 68 °C for 5 min. Constructs were sequenced completely to exclude any mutations.
Tab. 3.6 Oligonucleotides used for cloning of ASM from FLAG-N2 vector into pmCherry-N1 vector. ASM variant Primer Sequence 5’ -> 3’ ASM Forward TCAGATCTCGAGCTCAAGCTTGCCGCCATGCCCCGCTACGGAGC full-length Reverse CTCACCATGGTGGCGACCGGTACGCAAAACAGTGGCCTTGG variants ASM-6 Forward TCAGATCTCGAGCTCAAGCTTGCCGCCATGCCCCGCTACGGAGC Reverse CTCACCATGGTGGCGACCGGTACTTTGGTACACACGGTAACCTGC
ASM-7 Forward TCAGATCTCGAGCTCAAGCTTGCCGCCATGCCCCGCTACGGAGC Reverse CTCACCATGGTGGCGACCGGTACCGGGAACAAAAATTCATATTGAGAGAG
42 3. Material and Methods
3.2.2.3 Site-directed mutagenesis
The QuickChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to mutate ASM wild type sequence by PCR. Mutagenesis was done on ASM wild type- FLAG construct according to the manufacturer’s instructions with primers for different ASM mutations (tab. 3.7). Cycle parameters were as follows: initial denaturation at 95 °C for 1 min, 18 cycles at 95 °C for 50 sec, 60 °C for 50 sec, 68 °C for 6 min, and a final extension at 68 °C for 7 min. Constructs were sequenced completely to exclude any spontaneous further mutations.
Tab. 3.7 Oligonucleotides used for site-directed mutagenesis of ASM wild type-FLAG construct to generate different ASM variants. Construct Primer Sequence 5’ -> 3’ pASM_1522A_M381I_FLAGN2 Forward CCGCCTCATCTCTCTCAATATTAATTTTTGTTCCCGTGAGAAC Reverse GTTCTCACGGGAACAAAAATTAATATTGAGAGAGATGAGGCGG pASM_1522G_M381I_FLAGN2 Forward CCGCCTCATCTCTCTCAATATTAATTTTTGTTCCCGTGAGAAC Reverse GTTCTCACGGGAACAAAAATTAATATTGAGAGAGATGAGGCGG pASM_1522A_S508A_FLAGN2 Forward GAAACTACTCCAGGAGCGCTCACGTGGTCCTGGAC Reverse GTCCAGGACCACGTGAGCGCTCCTGGAGTAGTTTC pASM_1522G_S508A_FLAGN2 Forward GAAACTACTCCAGGAGCGCTCACGTGGTCCTGGAC Reverse GTCCAGGACCACGTGAGCGCTCCTGGAGTAGTTTC pASM_1522A_P325A_FLAGN2 Forward GGGTAACCATGAAAGCACAGCTGTCAATAGCTTCCCTCC Reverse GGAGGGAAGCTATTGACAGCTGTGCTTTCATGGTTACCC pASM_1522G_P325A_FLAGN2 Forward GGGTAACCATGAAAGCACAGCTGTCAATAGCTTCCCTCC Reverse GGAGGGAAGCTATTGACAGCTGTGCTTTCATGGTTACCC pASM_1522A_A487V_FLAGN2 Forward CTTCCTGGCACCCAGTGTAACTACCTACATCGGCC Reverse GGCCGATGTAGGTAGTTACACTGGGTGCCAGGAAG pASM_1522G_A487V_FLAGN2 Forward CTTCCTGGCACCCAGTGTAACTACCTACATCGGCC Reverse GGCCGATGTAGGTAGTTACACTGGGTGCCAGGAAG
3.2.2.4 PCR for detection of splice variants
For specific detection of different ASM splice variants, RT-PCR was carried out in a total reaction volume of 30 µl with 3 µl of undiluted cDNA, using 0.1 U/µl Tfi DNA polymerase with respective Tfi -buffer, 1.7 mM MgCl 2, 0.3 mM dNTPs (Invitrogen, Darmstadt, Germany) and 0.24 pmol/µl oligonucleotide primers (Operon, Ebersberg, Germany). The oligonucleotides and annealing temperatures used for ASM amplification are shown in table 3.8. Hypoxanthine-phosphoribosyl transferase (HPRT) encoding transcripts were amplified
43 3. Material and Methods as control (forward, 5’-TCCGCCTCCTCCTCTGCTC-3’; reverse, 5’-GAATAAACAC CCTTTCCAAATCCTCA-3’). Cycle parameters for all ASM-products were as follows: initial denaturation at 94 °C for 2 min, 35 cycles at 94 °C for 30 sec, ASM variant specific primer annealing temperature for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 3 min. Amplification products were analysed by agarose gel electrophoresis followed by ethidium bromide staining (Roth, Karlsruhe, Germany). A molecular weight standard (Generuler 100 bp plus DNA ladder; Fermentas, St. Leon-Rot, Germany) was used for size determination.
Tab. 3.8 Oligonucleotides used for amplification of ASM splice variants. ASM variant Primer Sequence 5’ -> 3’ Annealing temperature ASM-1 Forward CCTCAGAATTGGGGGGTTCTATGC 62.5 °C Reverse CACACGGTAACCAGGATTAAGG ASM-5 Forward TGCAGACCCACTGTGCTGC 66.5 °C Reverse GCCAGAAGTTCTCACG GGAACTG ASM-6 Forward CGTGAGAACTTCTGGCTCTTGATC 64.5 °C Reverse CTGCAAGGATGTGGGGCTGA ASM-7 Forward TGCAGACCCACTGTGCTGC 62.5 °C Reverse ACCCCCCAATTCTTTCTTTTCCC
3.2.2.5 Agarose gel electrophoresis
Using agarose gel electrophoresis DNA and RNA molecules can be identified and purified efficiently. Driven by voltage negatively charged molecules migrate to the positively charged anode, thereby being subject to separation by size. Ethidium bromide is incorporated into nucleic acids and fluoresces under UV light of 302 nm. For molecules larger than 1 kb a 1% agarose gel was used, for those smaller than 1 kb a 2% gel. Agarose was boiled and completely dissolved in 1x TBE buffer, cooled down to 50°C, and ethidium bromide (final concentration 0.5 µg/ml) was added. The dried gel was covered with TBE buffer and loaded with DNA or RNA mixed with 1x loading buffer (Fermentas, St. Leon-Rot, Germany). For size determination a molecular weight marker was loaded (Generuler 100 bp plus DNA ladder; Fermentas, St. Leon-Rot, Germany). Voltage of 5-10 V / cm was applied for 120 min, then nucleic acids within the gel were detected by UV light (302 nm).
44 3. Material and Methods
3.2.2.6 Cloning of ASM into FLAG-N2 expression vector
Purified PCR products of full-length ASM transcripts were cloned for analysis into p-SCB vector using StrataClone Blunt PCR cloning kit (Agilent, Waldbronn, Germany) according to manufacturer’s recommendations. For expression analysis, open reading frames were amplified by PCR from pSC-B-ASM plasmids. Purified PCR products were digested with restriction endonucleases Bsp E1 (recognition site T/CCGGA) and Bgl II (recognition site A/GATCT) (NEB, Frankfurt, Germany) and inserted between Xma I (recognition site C/CCGGG) and Bam HI (recognition site G/GATCC) sites within the multiple cloning site of the FLAG-N2 expression vector in frame with the coding sequence for the 16 amino acid FLAG-epitope (GTGDYKDDDDKRSLYK) (Fig. 3.1). In detail, vector DNA was digested with respective restriction endonucleases and purified. 100 ng linearized vector DNA was dephosphorylated using 1 U Shrimp Alkaline Phosphatase (Roche, Mannheim, Germany) at 37°C for 1 h, followed by an inactivation step at 65°C for 20 min. Ligation of DNA fragments, 100 ng vector DNA and 6-fold molar excess of insert DNA, was done using T4- Ligase (Invitrogen, Darmstadt, Germany) at 16°C for 24 h. After transformation of E. coli , positive clones were selected and DNA was isolated using QIAprep spin Miniprep kit (Qiagen, Hilden, Germany). Constructs were sequenced completely to exclude any mutations.
45 3. Material and Methods
Fig. 3.1 Plasmid construction for expression of ASM-FLAG constructs. ASM cDNA was amplified by PCR and inserted into FLAG-N2 expression vector via restriction endonuclease cleavage sites Bsp E1 and Bgl II in frame with the coding sequence for C-terminal FLAG-epitope.
3.2.2.7 Transformation of bacteria with DNA
For calcium chloride transformation 50 µl competent E. coli XL-1 Blue cells (Stratagene, La Jolla, CA, USA) were thawed and incubated with 10-50 ng plasmid DNA on ice. After heat shock of 45 sec at 42 °C cells were placed on ice for 2 min. Upon adding 450 µl SOC medium (37 °C) transformed bacteria were incubated on a shaker at 37 °C and 200 U/min for 60 min. 150 µl were plated on LB plates containing ampicillin (50 µg/ml) or kanamycin (100 µg/ml). Plates were incubated over night at 37 °C.
46 3. Material and Methods
3.2.2.8 Isolation of plasmid DNA
For isolating plasmid DNA 300 ml 2xYT medium containing kanamycin were incubated with a single colony of bacteria containing the respective plasmid. Bacteria were incubated over night at 37°C and shaken at 150U/min. By centrifugation at 4500g and 4°C for 20 min bacteria were pelleted. Preparation of plasmid DNA was carried out using Nucleobond xtra maxi EF (Macherey Nagel, Düren, Germany) according to manufacturer’s recommendations. Upon alkaline lysis of bacteria plasmid DNA is prepared by chromatography with a yield of 500-2000 µg DNA. Endotoxins were removed since plasmid DNA was used for cell culture experiments.
3.2.2.9 Sequencing of cDNA
Sequencing of cDNA was carried out according to the chain-termination method by Sanger (Sanger et al. , 1977) using Big Dye Mix (Perkin Elmer, Waltham, MA, USA) according to manufacturer’s recommendations. Following amplification programme was used: 96 °C for 30 sec; 50 °C for 5 sec; 60 °C for 4 min repeated in 30 cycles and final cycle 15 °C for 30 min. Primer are indicated in table 3.9. Analysis of sequencing was done by Lehrstuhl für Humangenetik, University Hospital Erlangen.
Tab. 3.9 Oligonucleotides used for sequencing of ASM plasmids. Primer Sequence (5` →3`) ASM binding site ASM_repeat_fw GGACTCCTTTGGATGGGCCT 85-104 ASM_repeat_rev CTTTGCAGATTGGGCAGG 269-286 huASM#2_fw AAGGAACCCAATGTGGCTCG 319-338 ASM_Exon2-1_fw TGCAGACCCACTGTGCTGC 669-687 huA-SMase_fw ATTGAGGGCAACCACTCCTC 1000-1030 NM_000543_BamH1_fw CACGGATCCCGCAGGAC 1194-1210
47 3. Material and Methods
3.2.3 RNA isolation and cDNA synthesis
3.2.3.1 RNA isolation
Total RNA was isolated from peripheral blood mononuclear cells, whole blood and from cell lines. PBMCs drawn from healthy volunteers were isolated from 30 ml of blood using Ficoll- density gradient centrifugation (Biocoll Separation Solution, Biochrom, Berlin, Germany). RNA from PBMCs and cell lines was extracted using Qiazol reagent (Qiagen, Hilden, Germany). 1 ml Qiazol was added to the cell pellet, which was sheared using a 20-gauge needle. After 5 min incubation at room temperature 200 µl chloroform were added and mixed. To separate RNA from DNA a centrifugation step was done at 13.000 rpm and 4°C for 15 min. The upper phase contains total RNA which was collected. To precipitate RNA 500 µl isopropanol were added and mixed. After 15 min incubation at room temperature RNA was pelleted by centrifugation at 13.000 rpm and 4°C for 10 min. Two washing steps were done using 75% ethanol, and the dried RNA pellet was resuspended in H 2O. For complete resolving RNA was incubated at 55°C for 10 min. RNA of depressed patients was isolated from whole blood within the ADT-study conducted at the Department of Psychiatry and Psychotherapy. RNA isolation of whole blood was done using PAXgene blood RNA kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. The concentration of RNA was determined photometrically (Nanodrop, Peqlab, Erlangen, Germany) at 260 nm, and RNA integrity was assessed by non-denaturing agarose gel electrophoresis and capillar gel electrophoresis (Experion, BioRad, Munich, Germany). The collection of blood samples was approved by the local Ethics Committee and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.
48 3. Material and Methods
3.2.3.2 Synthesis of cDNA
Synthesis of cDNA was carried out in a total reaction volume of 20 µl with 1 µg total RNA using the VILO cDNA reaction kit according to manufacturer’s recommendations (Invitrogen, Darmstadt, Germany).
3.2.4 Protein-biochemical analyses
3.2.4.1 Determination of protein concentration
The protein concentration of cell extracts was determined using the BCA assay (BioRad, Munich, Germany) according to manufacturer’s recommendations.
3.2.4.2 SDS-PAGE and Western blot analysis
Proteins were separated by polyacrylamide gel electrophoresis under denaturing conditions in a minigel chamber (Mini Protein 3 cell, BioRad, Munich, Germany). Sample aliquots were denatured in SDS-running buffer for 5 min. Total protein (10 µg) was separated by 10% sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was composed of a 4% acrylamide stacking gel (upper SDS gel) and a 10% acrylamide separating gel (lower SDS gel):
Lower SDS gel (10 % T) 4x Lower gel buffer (1.5 M Tris-HCl, pH 8.8) 2.5 ml 30%T/2.6%C Acrylamide 3.3 ml 10% SDS 0.1 ml
H20bidest 4.02 ml 10% APS 40 µl TEMED 10 µl
49 3. Material and Methods
Upper SDS gel (4 % T) 4x Upper gel buffer (0.5 M Tris-HCl, pH 6.8) 1.25 ml 30%T/2.6%C Acrylamide 0,667 ml 10% SDS 50 µl
H20bidest 2,973 ml 10% APS 40 µl TEMED 10 µl
For controlling gel electrophoresis a pre-stained molecular weight standard was loaded (Page Ruler Prestained Protein Ladder, Fermentas, St. Leon-Rot, Germany), for determining the size of proteins an unstained molecular weight standard (Magic Mark, Invitrogen, Darmstadt, Germany). Gel electrophoresis was performed in Laemmli buffer (Laemmli, 1970) at 120 V for about 2 h.
Separated proteins were transferred to a PVDF- membrane (Millipore, Schwalbach/Ts, Germany) by semi-dry blotting (SemiPhor Transfer Unit TE77, Amersham, Munich, Germany) at 100 mA for 1 h using Towbin buffer.
Blots were washed with PBS/ T and incubated in 2.5% milk powder (Roth, Karlsruhe, Germany) and 0.1% Tween 20 (Sigma-Aldrich, Munich, Germany) in PBS overnight at 4 °C to reduce unspecific binding. After washing with PBS/0.1% Tween 20 for 15 minutes, blots were incubated for 4 h at room temperature with primary monoclonal FLAG-antibody derived from mouse (1:1000; Sigma-Aldrich, Munich, Germany), and for 1 hour with primary Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody from mouse (1:200.000; Millipore, Schwalbach/Ts, Germany). After washing in PBS/0.1% Tween 20 three times, blots were incubated for 1 hour at room temperature with secondary goat anti- mouse IgG antibody, coupled to horseradish peroxidase (1:10.000; Dianova, Hamburg, Germany). All antibodies were diluted in 0.5% milk powder solution (Roth, Karlsruhe, Germany) and 0.1% Tween 20 in PBS (Sigma-Aldrich, Munich, Germany). Detection was performed using freshly mixed enhanced chemiluminescence developer reagent (ECL, Amersham, Munich, Germany). After incubation of 5 min signals were visualized on a high- sensitivity camera device (FluorSMax, BioRad, Munich, Germany).
50 3. Material and Methods
3.2.4.3 In vitro determination of ASM activity
Quantitative analysis of ASM activity was performed on 10 µg of total cellular protein extract, diluted in 250 µl of lysis buffer; 440 pmol choline methyl-14 C sphingomyelin (Perkin-Elmer, Waltham, MA, USA) was suspended in 30 µl of enzyme buffer [250 mM sodium acetate pH 5.0 (Merck, Darmstadt, Germany), 0.1% NP-40 (Sigma-Aldrich, Munich, Germany), 1.3 mM EDTA (Sigma-Aldrich, Munich, Germany)] and added to the protein extract. The enzymatic reaction was incubated at 37 °C for 20 min. The reaction was stopped by addition of 800 µl of chloroform/methanol (2:1), and phases were separated by centrifugation. Radioactivity of the 14 C-labelled product phosphorylcholine in the aqueous phase was determined by liquid scintillation counting and allowed quantification of ASM activity. Results are given as ASM activity in nmol/mg/h and refer to the mean values resulting from three independent experiments. Alternatively, the activity of ASM was determined using the fluorescent substrate BODIPY- C12-sphingomyelin (Invitrogen, Darmstadt, Germany). The reaction consisted of 116 pmol substrate in 200 mM sodium acetate buffer (pH 5.0) with 500 mM NaCl and 0.2% NP-40 in a total volume of 100 µl and was started by the addition of 2 µl cell lysate typically corresponding to 0.5-2 µg protein. After incubation for 0.5 to four hours at 37°C, the fluorescent product ceramide and un-cleaved substrate were extracted by addition of 250 µl chloroform/methanol (2:1). Following vortexing and centrifugation, the lower phase was concentrated in a SpeedVac vacuum concentrator and spotted on silica gel 60 plates (Merck, Darmstadt, Germany). Ceramide and sphingomyelin were separated by thin layer chromatography using chloroform/methanol (80:20) as a solvent and quantified on a Typhoon Trio scanner (GE Healthcare, 488 nm excitation, 520 nm emission, 280 V, 100 µm resolution) with the QuantityOne software (BioRad, Munich, Germany). Enzymatic activity of ASM was calculated as hydrolysis rate of sphingomyelin to ceramide per time and per protein of the cell lysate sample (pmol/µg/h).
51 3. Material and Methods
3.2.4.4 MALDI-TOF MS analysis of cellular ceramide and sphingomyelin levels
After thawing, cell samples were homogenized and sonicated. Lipids were subjected to classical chloroform/methanol extraction (Folch et al. , 1957), transferred to a clean tube, dried, and resolved (2% w/v) in chloroform. For MALDI-TOF MS, samples were mixed with matrix consisting of 0.5 M 2.5-dihydroxybenzoic acid in methanol with 0.1% trifluoroacetic acid (spectrometric grade, Sigma-Aldrich, Munich, Germany) (Schiller et al. , 2004). Aliquots were spotted on a steel target and subjected to mass spectrometric analysis. Mass spectra were obtained using an Autoflex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser ( λ= 337 nm). Mass spectra were acquired in reflectron and positive ion mode. Each spectrum consists of the average of 100 laser shots. Obtained mass-to-charge ratio (m/z) values were compared to the lipid database LIPID MAPS (http://www.lipidmaps.org), according to which relevant corresponding ceramide and sphingomyelin species were defined for the semi-quantitative analysis (Löhmann et al. ). The relative intensities of both mass signals [M+H ]+ and [M+Na ]+ or [M+K ]+, respectively, were determined and summarized. The ratio of ceramide to sphingomyelin intensities was calculated for each individual spectrum and averaged over three individual samples.
3.2.5 In silico analyses and statistics
In silico analyses of ASM orthologous transcripts were performed using the NCBI database GenBank. Alignments were generated with the ClustalW alignment of the European Bioinformatics Institute (www. ebi.ac.uk/clustalw). The molecular weight of the discussed proteins was calculated using the molecular weight calculator at www.sciencegateway.org/ tools/proteinmw.htm. Images were edited with Adobe Photoshop CS5. For statistical analysis GraphPad-Prism 4.0 software (GraphPad Software Inc., La Jolla) was used.
52 4. Results
4. Results
4.1 Impact of DNA sequence variations on Acid Sphingomyelinase activity
For investigating effects of DNA sequence variations on properties of the ASM enzyme, we cloned five different ASM missense variations. To establish a model system for validating the cell-biological methods, we used ASM variants with already published characteristics. For this purpose we controlled properties of ASM wild type (WT), the ASM variant c.1528T>G / p.S508A, which was recently investigated (Jenkins et al. , 2010a; Jenkins et al. , 2010b), and the Niemann-Pick disease type A variant c.1146G>T / p.M381I, described by Takahashi et al. (1992b). ASM sequencing of 58 patients suffering from major depression revealed three ASM missense variations: Variation c.973C>G / p.P325A is located within the region coding for the catalytic domain, and variations c.1460C>T / p.A487V and c.1522G>A / p.G506R are located in the C-terminal coding region of ASM (Fig. 4.1). These ASM variants should be characterized.
Fig. 4.1 Position of ASM variations investigated in this study. Five different ASM variants were characterized. ASM wild type and ASM variants p.S508A and p.M381I served as model system. ASM variations located within the catalytic domain (p.P325A) and the C-terminal domain (p. A487A, p.G508R) were under investigation. Scheme is drawn to scale.
In our approach, we cloned these different ASM variations and transiently expressed the resulting ASM variants in a cell culture system. Due to the putative N-terminal signal peptide, we expressed ASM variants as C-terminal epitope-tagged fusion proteins. First of all, we cloned cDNA of ASM wild type derived from RNA of a healthy volunteer into the
53 4. Results expression vector FLAG-N2 for protein-biochemical analyses. For a better tracking of ASM variants in localization studies, we sub-cloned ASM wild type into expression vector pmCherry-N1, which contains the sequence for a red fluorescent protein (Fig. 4.2). The resulting ASM-FLAG and ASM-Cherry constructs were sequenced to confirm their integrity.
Fig. 4.2 Cloning approach for the generation of recombinant ASM constructs containing different ASM variations and FLAG- or Cherry-epitope. Starting from direct cloning of donor cDNA into expression vector FLAG-N2, all further ASM constructs were obtained by mutagenesis or infusion cloning, respectively.
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4.1.1 Expression of recombinant ASM variants
To characterize the different ASM variants biochemically, ASM-FLAG variants were transiently expressed in HeLa cells. Expression of ASM-FLAG variants was confirmed by Western blot analyses 48 h upon transfection using FLAG antibody. Detection of the housekeeping protein GAPDH served as loading control. Analyses showed that all cloned ASM variants were translated into protein and displayed predicted sizes. Lysates derived from vector-transfected cells served as negative control and did not result in detectable signals (Fig. 4.3).
Fig. 4.3 All investigated ASM variants are translated into protein and display predicted sizes. ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were analysed by Western blotting 48 h after transfection. Expression was detected using FLAG antibody. Lysates of vector-transfected cells served as negative control, GAPDH was detected for loading control. Indicated are representative Western blot analyses of three independent experiments. M indicates size marker.
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Of note, protein expression levels differed highly between ASM variants with weakest expression of p.G506R/S508A (27% of WT expression) and strongest expression of p.G506R/M381I and p.M381I (309% and 290% of WT expression) (Fig. 4.4). To account for expression differences, all further biochemical analyses were corrected for respective expression levels.
Fig. 4.4 ASM variants were transiently expressed to a different extent. Protein expression of ASM p.G506R/S508A was reduced to 27% of WT expression, whereas ASM p.G506R/M381I and p.M381I were three-fold stronger expressed than ASM WT. Other variants reached values between these extremes. ASM-FLAG constructs were transiently expressed in HeLa cells. Expression of each ASM variant was verified by Western blot analyses. Signals derived from immunoblot band intensities were quantified using QuantityOne software (BioRad, Munich, Germany). Expression of ASM variants is given in percentage expression relative to transient expression of ASM WT (100%). Band intensities of transiently expressed ASM variants were normalized to the respective GAPDH signal. Displayed data represent mean values of three independent experiments, error bars represent SD.
4.1.2 Characterization of ASM variants used for model system
For validating the methodical approach, a model system composed of three different ASM variants was analysed. The ASM wild type is described as the catalytic active enzyme located inside the lysosome. The artificial ASM variant p.S508A was characterized as an ASM variant exerting about 30-60% of enzymatic wild type activity with lysosomal localization. Changing the phosphorylation site p.508 by the substitution of serine by alanine is shown to
56 4. Results affect activation of ASM and seems to be crucial for secretion of ASM (Jenkins et al. , 2010a; Jenkins et al. , 2010b). The Niemann-Pick type A variant p.M381I was described by Takahashi et al. and is characterized by an exchange of methionin by isoleucin, both being apolar amino acids. This variant did not exert any catalytic activity (Takahashi et al. , 1992b) and should be trapped inside the ER like Niemann-Pick variants according to Jenkins et al. (2010b).
4.1.2.1 Catalytic activity of ASM model system variants
In our approach, the intrinsic activity of different ASM variants was determined upon in vitro over-expression. ASM-FLAG constructs were transiently expressed in HeLa cells. Cells were lysed 48 h upon transfection and ASM activity of crude cell lysates was analysed using a radioactive enzyme-activity-assay (Gulbins and Kolesnick, 2000). Over-expression of ASM wild type elevated ASM activity averaged 35-fold with respect to endogenous ASM activity levels which were controlled by vector control conditions (223 nmol/mg/h vs. 6.4 nmol/mg/h). For evaluating ASM variants, ASM wild type activity was set on 100%. Due to differential protein expression levels, exogenous enzymatic activity was normalized according to quantified Western blot signals and corrected for loading variations. Therefore, enzymatic activity of each investigated ASM variant was given in exogenous ASM activity per expressed protein with respect to ASM wild type activity. Compared to ASM wild type, ASM variant p.S508A exerted an enzymatic activity of 33% (SD ± 2.3), whereas Niemann-Pick variant p.M381I was catalytically inactive. The difference between ASM wild type and model systems variants was statistically significant (Fig. 4.5). Enzymatic activities of our ASM model system variants therefore reveal similar levels of ASM activity as described earlier. The ASM variants wild type, p.S508A and p.M381I thus qualify this established method for biochemical analyses of further ASM variants.
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Fig. 4.5 ASM model system variants display predicted enzymatic activity levels. Compared to ASM wild type, ASM variants p.S508A and p.M381I exerted 33% (SD ± 2.3) and 0% catalytic activity, respectively. ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzyme-activity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in exogenous ASM activity levels per expressed protein in percentage relative to activity of ASM WT (100%). Displayed data represent mean values of three independent experiments, error bars represent SD. Statistical significance was calculated with respect to WT (* p<0.05).
4.1.2.2 Localization of ASM model system variants
With the aim to track the localization of ASM variants we investigated the subcellular localization of ASM-Cherry constructs, since the red fluorescent tag allows easy detection. H4 cells were transiently transfected with ASM-Cherry constructs of ASM wild type and ASM variants p.S508A and p.M381I. After 48 h cells were fixed and immunostained for the lysosomal marker protein LAMP1 and the endoplasmic reticulum marker protein PDIA4 for determining localization of lysosomes and endoplasmic reticulum, respectively. Cells were analysed for their subcellular localization pattern using fluorescence microscopy and for potential co-localization with lysosomes or endoplasmic reticulum using confocal microscopy.
ASM wild type localized within a dotted subcellular pattern. Also ASM variant p.S508A was found to occur within dotted structures, in sharp contrast to p.M381I (Fig. 4.6).
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Fig. 4.6 Subcellular localization patterns of ASM model system variants ASM wild type and ASM variants p.S508A and p.M381I. ASM wild type (A) and p.S508A (B) localized in dotted subcellular patterns, whereas p.M381I (C) did not. ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are representative cells investigated by fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three times.
Confocal microscopy revealed that signals of ASM-Cherry wild type construct and LAMP1 were co-localized. Therefore, ASM wild type was found to be localized within lysosomal compartments. Also p.S508A showed co-localization with lysosomal marker protein LAMP1 (Fig. 4.7 A). In contrast, p.M381I showed co-localization with endoplasmic reticulum marker PDIA4 (Fig. 4.7 B).
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Fig. 4.7 Co-localization of ASM model system variants with subcellular organelles. ASM wild type and ASM variant p.S508A were localized within lysosomes, ASM variant p.M381I within the endoplasmic reticulum. Confocal microscopy revealed co-localization of ASM wild type and p.S508A with lysosomal marker protein LAMP1 (A) and of ASM variant p.M381I with endoplasmic reticulum marker PDIA4 (B). ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Marker proteins were detected by immunofluorescence with anti-LAMP1 and anti-PDIA4 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASM- Cherry constructs in red, marker proteins in green and merged signals. The indicated scale covers 10 µm. Co- localization experiments were reproduced three times.
Compared to already published localization studies, ASM-Cherry wild type construct displayed identical localization patterns as did endogenous ASM wild type (Jones et al. , 2008) and as described for ASM in different over-expression studies (Ni and Morales, 2006; Jin et al. , 2008). This indicates that the C-terminal Cherry-epitope does not influence proper localization of ASM inside the lysosome. Also published data about localization of ASM variant p.S508A and predicted localization of ASM variant p.M381I match our results concerning localization within lysosomes or endoplasmic reticulum, respectively (Jenkins et al. , 2010a; Jenkins et al. , 2010b).
4.1.3 Characterization of ASM variant p.P325A
The rare ASM variant p.P325A shows an amino acid exchange within the catalytic domain of ASM. The apolar amino acid proline is substituted by the apolar amino acid alanine. This variant was also detected in patients suffering from Niemann-Pick disease type B (Simonaro et al. , 2002).
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4.1.3.1 ASM variant p.P325A is catalytically inactive
Catalytic activity of ASM variant p.P325A was determined analogous to the approach of determining enzymatic activity of ASM variants used for our model system. Compared to ASM wild type, ASM variant p.P325A did not exert catalytic activity (Fig. 4.8).
Fig. 4.8 ASM variant p.P325A does not exert catalytic activity compared to ASM wild type. ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzyme- activity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in exogenous ASM activity levels per expressed protein in percentage relative to activity of ASM WT (100%). Displayed data represent mean values of three independent experiments, error bars represent SD.
4.1.3.2 ASM variant p.P325A localizes in lysosomes
With the purpose to determine the subcellular organelles in which ASM variant p.P325A localizes, we conducted localization analyses as described in 4.1.2.2. ASM variant p.P325A localized within a dotted subcellular pattern when investigated by fluorescence microscopy. Compared to ASM wild type, patterns were highly similar (Fig. 4.9).
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Fig. 4.9 ASM variant p.P325A localizes in a dotted subcellular pattern. ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are representative cells investigated by fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three times.
Signals of ASM-Cherry variant p.P325A and LAMP1 were co-localized as revealed by confocal microscopy. Thus, ASM variant p.P325A was localized within the lysosome (Fig. 4.10).
Fig. 4.10 ASM variant p.P325A is localized within lysosomes. Confocal microscopy revealed co-localization of ASM-Cherry variant p.P325A with lysosomal marker protein LAMP1. The ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Lysosomal marker protein was detected by immunofluorescence with anti-LAMP1 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASM- Cherry construct in red, marker protein in green and merged signals. The indicated scale covers 10 µm. Co- localization experiments were reproduced three times.
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4.1.4 Characterization of ASM variant p.A487V
ASM variant p.A487V shows an amino acid exchange within the C-terminal domain of ASM. Alanine is substituted by valine, both being apolar amino acids. Also patients suffering from Niemann-Pick disease type B displayed this ASM variation (Simonaro et al. , 2002).
4.1.4.1 Catalytic activity of ASM variant p.A487V is equivalent to wild type
Enzymatic activity of ASM variant p.A487V was determined as described in 4.1.2.1. ASM variant p.A487V displayed 108 % (SD ± 63.1) of ASM wild type activity, indicating similar ASM activity levels of both ASM enzymes (Fig. 4.11).
Fig. 4.11 Enzymatic activities of ASM variant p.A487V and ASM wild type reach similar levels. ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzyme- activity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in exogenous ASM activity levels per expressed protein in percentage relative to activity of ASM WT (100%). Displayed data represent mean values of three independent experiments, error bars represent SD.
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4.1.4.2 ASM variant p.A487V localizes in lysosomes
As described for the other variants, localization of ASM variant p.A487V was investigated using the ASM-Cherry construct, which localized in a dotted subcellular pattern (Fig. 4.12).
Fig. 4.12 ASM variant p.A487V localizes in a dotted subcellular pattern. ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are representative cells investigated by fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three times.
Signals of ASM-Cherry variant p.A487V and LAMP1 were co-localized as revealed by confocal microscopy, which means a lysosomal localization of ASM variant p.A487V (Fig. 4.13).
Fig. 4.13 ASM variant p.A487V localizes within lysosomes. Confocal microscopy revealed co-localization of ASM variant p.A487A with the lysosomal marker protein LAMP1. The ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Lysosomal marker protein was detected by immunofluorescence with anti-LAMP1 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASM- Cherry construct in red, marker protein in green and merged signals. The indicated scale covers 10 µm. Co- localization experiments were reproduced three times.
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4.1.5 Characterization of ASM variant p.G506R
The common ASM variation p.G506R is characterized by an amino acid exchange within the C-terminal domain of ASM, located two amino acids next to the phosphorylation site p.508. Due to this close proximity, biochemical effects were supposed. The apolar amino acid glycine is replaced by the polar and positively charged amino acid arginine. To get insight into the potential effect of this variation, we cloned the variant p.G506R not only as single substitution, but also in combination with the so far presented variations p.S508A, p.M381I, p.P325A and p.A487V.
4.1.5.1 ASM variation p.G506R impacts catalytic activity
For determining the effect of ASM variation p.G506R on catalytic activity, we considered eight different variants, resulting in four pairs: G506R - WT; G506R/S508A - S508A; G506R/M381I - M381I; G506R/P325A - P325A; G506R/A487V - A487V. Determination of enzymatic activity of ASM variants was described in 4.1.2.1. The singular variation p.G506R showed a statistically significant decrease in ASM activity of 25% compared to wild type (80% ± 12.7 vs. 100% of wild type activity; p<0.05). Also regarding variant p.A487V, the combination with variation p.G506R resulted in a decrease in ASM activity of 37% (71% ± 20.1 vs. 108% ± 63.1 of wild type activity). In contrast, nearly no change in ASM activity was detected between combined variant p.G506R/S508A in comparison with p.S508A (43% ± 18.1 and 33% ± 2.3 of wild type activity). No enzymatic activity was detected for combined variants p.G506R/M381I and p.G506R/P325A as described for p. M381I and p.P325A (Fig. 4.14). The variation p.S508A not only made a statistically significant decrease in ASM activity when occurring singularly (p.S508A compared to wild type, see also 4.1.2.1), but also when combined with variation p.G506R (p.G506R - 80% ± 12.7 vs. p.G506R/S508A - 44% ± 18 of wild type activity; p<0.05) (Fig. 4.14).
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Fig. 4.14 ASM variation p.G506R causes a decrease in enzymatic activity. Compared to wild type, variation p.G506R lead to a 25%-decrease in ASM activity. The combination of p.G506R with a second variation as described above made differential effects on ASM activity observable: For variant p.A487V a 37%-decrease, for p.S508A only a slight effect of 10% and for catalytically inactive variants p.M381I and p.P325A no effect on enzymatic activity was detected. ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzyme-activity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in exogenous ASM activity levels per expressed protein in percentage relative to activity of ASM WT (100%). Displayed data represent mean values of three independent experiments, error bars represent SD. Statistical significance was calculated by one way ANOVA (* p<0.05).
4.1.5.2 ASM variation p.G506R impacts localization
Analogous to ASM variants described above, subcellular localization of the ASM variants containing ASM variation p.G506R was investigated. ASM variants p.G506R, p.G506R/P325A and p.G506R/A487V localized in dotted subcellular patterns, in contrast to p.G506R/S508A and p.G506R/M381I (Fig. 4.15). Compared to the singular occurrence of these variations (see above), the combination with variation p.G506R only makes a difference in localization patterns for ASM variation S508A (p.S508A - dotted structure, p.G506R/S508A - non-dotted structure).
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Fig. 4.15 Subcellular localization patterns of ASM variants containing the variation p.G506R. ASM variants p.G506R (A) , p.G506R/P325A (D) and p.G506R/A487V (E) localized in dotted subcellular patterns, in contrast to p.G506R/S508A (B) and p.G506R/M381I (C) . ASM-Cherry constructs (p.G506R, p.G506R/S508A and p.G506R/M381I) were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). ASM-FLAG constructs (p.G506R/P325A and p.G506R/A487V) were detected by immunofluorescence with anti-FLAG primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three times.
Using confocal microscopy we detected that signals of ASM-Cherry construct p.G506R and the lysosomal marker protein LAMP1 were co-localized, indicating lysosomal localization (Fig. 4.16 A). Signals of ASM variants p.G506R/S508A and p.G506R/M381I showed co- localization with signals derived from endoplasmic reticulum marker PDIA4, which indicates that these variants were located within the endoplasmic reticulum (Fig. 4.16 B). For technical reasons we did not investigate co-localization of ASM variants p.G506R/P325A and p.G506R/A487V.
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Fig. 4.16 Co-localization of ASM variants containing variation p.G506R with subcellular organelles. ASM variant p.G506R was localized within lysosomes, ASM variants p.G506R/S508A and p.G506R/M381I within the endoplasmic reticulum. Confocal microscopy revealed co-localization of ASM variant p.G506R with lysosomal marker protein LAMP1 (A) and of ASM variant p.G506R/S508A and p.G506R/M381I with endoplasmic reticulum marker PDIA4 ( B). ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Marker proteins were detected by immunofluorescence with anti-LAMP1 and anti- PDIA4 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASM-Cherry constructs in red, marker proteins in green and merged signals. The indicated scale covers 10 µm. Co-localization experiments were reproduced three times.
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4.1.6 Summary: Characterization of ASM variants
Investigating catalytic activity and subcellular localization reveals differential properties of ASM variants. Five different ASM variations were cloned and transiently expressed in a cell culture system. Concerning catalytic activity, ASM variants can be divided in three different categories: full (p.A487V), reduced (p.S508A, p.G506R) or no catalytic activity (p.M381I, p.P325A). Concerning subcellular localization, ASM variants build two different groups: lysosomal (p.S508A, p.P325A, p.A487V, p.G506R) or non-lysosomal (p.M381I, p.G506R/S508A) localization. A short overview of differential properties of ASM variants is given in table 4.1. To sum up, most of ASM variations detected also within depressed patients showed differential characteristics, but they did not cause excessive ASM activity.
Tab. 4.1 ASM variants display differential properties. Enzymatic Protein Nucleotide activity expression ASM variant Localization change [% wild type] [% wild type] ± SD ±SD ASM wild type - 100 100 Lysosome
p.S508A c.1528T>G 34±2 144±26 Lysosome
p.M381I c.1146G>A 0±1 290±115 ER
p.P325A c.973C>G 0±1 181±64 Lysosome
p.A487V c.1460C>T 109±63 165±67 Lysosome
p.G506R c.1522G>A 76±13 99±13 Lysosome
p.G506R/ c.1522G>A 44±18 27±6 ER S508A c.1528T>G p.G506R/ c.1522G>A 0±1 309±119 ER M381I c.1146G>A p.G506R/ c.1522G>A 0±0 228±81 n.d. P325A c. c.973C>G p.G506R/ c.1522G>A 72±20 170±61 n.d. A487V c.1460C>T
Summarized characteristics of ASM variants. Enzymatic activity indicates mean values ± SD (n=3) of exogenous ASM activity per expressed protein in % of WT activity. Protein expression indicates mean values ± SD (n=3) of ASM band intensities per µg protein controlled by GAPDH band intensities in % of WT values. For p.G506R/P325A and p.G506R/A487V no co-localization studies were conducted (n.d.).
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4.2 Impact of alternative splicing on Acid Sphingomyelinase activity
In the attempt to clone variant full-length ASM transcripts for expression studies, we isolated total human RNA from different sources and synthesized the corresponding cDNAs. The complete ASM coding sequence was amplified by RT-PCR using primers flanking the ATG initiation codon and the stop codon, inserted into a vector and sequenced. In total, 17 transcripts from two different sources were isolated and analysed, of which 13 transcripts were identical to the reference sequence of ASM with the exception of known polymorphisms (Schuchman et al. , 1991a; Wan and Schuchman, 1995). Sequence alignment of four transcripts, however, revealed unique local differences that most probably arose from alternative splicing (Fig. 4.17).
ASM-1 ATGCCCCGCTACGGAGCGTCACTCCGCCAGAGCTGCCCCAGGTCCGGCCGGGAGCAGGGA 60 ASM-5 ATGCCCCGCTACGGAGCGTCACTCCGCCAGAGCTGCCCCAGGTCCGGCCGGGAGCAGGGA 60 ASM-6 ATGCCCCGCTACGGAGCGTCACTCCGCCAGAGCTGCCCCAGGTCCGGCCGGGAGCAGGGA 60 ASM-7 ATGCCCCGCTACGGAGCGTCACTCCGCCAGAGCTGCCCCAGGTCCGGCCGGGAGCAGGGA 60 ************************************************************
ASM-1 CAAGACGGGACCGCCGGAGCCCCCGGACTCCTTTGGATGGGCCTGGTGCTGGCGCTGGCG 120 ASM-5 CAAGACGGGACCGCCGGAGCCCCCGGACTCCTTTGGATGGGCCTGGTGCTGGCGCTGGCG 120 ASM-6 CAAGACGGGACCGCCGGAGCCCCCGGACTCCTTTGGATGGGCCTGGTGCTGGCGCTGGCG 120 ASM-7 CAAGACGGGACCGCCGGAGCCCCCGGACTCCTTTGGATGGGCCTGGCGCTGGCGCTGGCG 120 ********************************************** *************
ASM-1 CTGGCGCTGGCGCTGGCGCTGGCTCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG 180 ASM-5 CTGGCGCTGGCGCTGGC------TCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG 174 ASM-6 CTGGCGCTGGCGCTGGC------TCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG 174 ASM-7 CTGGCGCTGGC------TCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG 168 *********** *************************************
ASM-1 GCTCACCCTCTTTCTCCCCAAGGCCATCCTGCCAGGTTACATCGCATAGTGCCCCGGCTC 240 ASM-5 GCTCACCCTCTTTCTCCCCAAGGCCATCCTGCCAGGTTACATCGCATAGTGCCCCGGCTC 234 ASM-6 GCTCACCCTCTTTCTCCCCAAGGCCATCCTGCCAGGTTACATCGCATAGTGCCCCGGCTC 234 ASM-7 GCTCACCCTCTTTCTCCCCAAGGCCATCCTGCCAGGTTACATCGCATAGTGCCCCGGCTC 228 ************************************************************
ASM-1 CGAGATGTCTTTGGGTGGGGGAACCTCACCTGCCCAATCTGCAAAGGTCTATTCACCGCC 300 ASM-5 CGAGATGTCTTTGGGTGGGGGAACCTCACCTGCCCAATCTGCAAAGGTCTATTCACCGCC 294 ASM-6 CGAGATGTCTTTGGGTGGGGGAACCTCACCTGCCCAATCTGCAAAGGTCTATTCACCGCC 294 ASM-7 CGAGATGTCTTTGGGTGGGGGAACCTCACCTGCCCAATCTGCAAAGGTCTATTCACCGCC 288 ************************************************************
ASM-1 ATCAACCTCGGGCTGAAGAAGGAACCCAATGTGGCTCGCGTGGGCTCCGTGGCCATCAAG 360 ASM-5 ATCAACCTCGGGCTGAAGAAGGAACCCAATGTGGCTCGCGTGGGCTCCGTGGCCATCAAG 354 ASM-6 ATCAACCTCGGGCTGAAGAAGGAACCCAATGTGGCTCGCGTGGGCTCCGTGGCCATCAAG 354 ASM-7 ATCAACCTCGGGCTGAAGAAGGAACCCAATGTGGCTCGCGTGGGCTCCGTGGCCATCAAG 348 ************************************************************
ASM-1 CTGTGCAATCTGCTGAAGATAGCACCACCTGCCGTGTGCCAATCCATTGTCCACCTCTTT 420 ASM-5 CTGTGCAATCTGCTGAAGATAGCACCACCTGCCGTGTGCCAATCCATTGTCCACCTCTTT 414 ASM-6 CTGTGCAATCTGCTGAAGATAGCACCACCTGCCGTGTGCCAATCCATTGTCCACCTCTTT 414 ASM-7 CTGTGCAATCTGCTGAAGATAGCACCACCTGCCGTGTGCCAATCCATTGTCCACCTCTTT 408 ************************************************************
ASM-1 GAGGATGACATGGTGGAGGTGTGGAGACGCTCAGTGCTGAGCCCATCTGAGGCCTGTGGC 480 ASM-5 GAGGATGACATGGTGGAGGTGTGGAGACGCTCAGTGCTGAGCCCATCTGAGGCCTGTGGC 474 ASM-6 GAGGATGACATGGTGGAGGTGTGGAGACGCTCAGTGCTGAGCCCATCTGAGGCCTGTGGC 474 ASM-7 GAGGATGACATGGTGGAGGTGTGGAGACGCTCAGTGCTGAGCCCATCTGAGGCCTGTGGC 468 ************************************************************
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ASM-1 CTGCTCCTGGGCTCCACCTGTGGGCACTGGGACATTTTCTCATCTTGGAACATCTCTTTG 540 ASM-5 CTGCTCCTGGGCTCCACCTGTGGGCACTGGGACATTTTCTCATCTTGGAACATCTCTTTG 534 ASM-6 CTGCTCCTGGGCTCCACCTGTGGGCACTGGGACATTTTCTCATCTTGGAACATCTCTTTG 534 ASM-7 CTGCTCCTGGGCTCCACCTGTGGGCACTGGGACATTTTCTCATCTTGGAACATCTCTTTG 528 ************************************************************
ASM-1 CCTACTGTGCCGAAGCCGCCCCCCAAACCCCCTAGCCCCCCAGCCCCAGGTGCCCCTGTC 600 ASM-5 CCTACTGTGCCGAAGCCGCCCCCCAAACCCCCTAGCCCCCCAGCCCCAGGTGCCCCTGTC 594 ASM-6 CCTACTGTGCCGAAGCCGCCCCCCAAACCCCCTAGCCCCCCAGCCCCAGGTGCCCCTGTC 594 ASM-7 CCTACTGTGCCGAAGCCGCCCCCCAAACCCCCTAGCCCCCCAGCCCCAGGTGCCCCTGTC 588 ************************************************************
ASM-1 AGCCGCATCCTCTTCCTCACTGACCTGCACTGGGATCATGACTACCTGGAGGGCACGGAC 660 ASM-5 AGCCGCATCCTCTTCCTCACTGACCTGCACTGGGATCATGACTACCTGGAGGGCACGGAC 654 ASM-6 AGCCGCATCCTCTTCCTCACTGACCTGCACTGGGATCATGACTACCTGGAGGGCACGGAC 654 ASM-7 AGCCGCATCCTCTTCCTCACTGACCTGCACTGGGATCATGACTACCTGGAGGGCACGGAC 648 ************************************************************
ASM-1 CCTGACTGTGCAGACCCACTGTGCTGCCGCCGGGGTTCTGGCCTGCCGCCCGCATCCCGG 720 ASM-5 CCTGACTGTGCAGACCCACTGTGCTGCCGCCGGGGTTCTGGCCTGCCGCCCGCATCCCGG 714 ASM-6 CCTGACTGTGCAGACCCACTGTGCTGCCGCCGGGGTTCTGGCCTGCCGCCCGCATCCCGG 714 ASM-7 CCTGACTGTGCAGACCCACTGTGCTGCCGCCGGGGTTCTGGCCTGCCGCCCGCATCCCGG 708 ************************************************************
ASM-1 CCAGGTGCCGGATACTGGGGCGAATACAGCAAGTGTGACCTGCCCCTGAGGACCCTGGAG 780 ASM-5 CCAGGTGCCGGATACTGGGGCGAATACAGCAAGTGTGACCTGCCCCTGAGGACCCTGGAG 774 ASM-6 CCAGGTGCCGGATACTGGGGCGAATACAGCAAGTGTGACCTGCCCCTGAGGACCCTGGAG 774 ASM-7 CCAGGTGCCGGATACTGGGGCGAATACAGCAAGTGTGACCTGCCCCTGAGGACCCTGGAG 768 ************************************************************
ASM-1 AGCCTGTTGAGTGGGCTGGGCCCAGCCGGCCCTTTTGATATGGTGTACTGGACAGGAGAC 840 ASM-5 AGCCTGTTGAGTGGGCTGGGCCCAGCCGGCCCTTTTGATATGGTGTACTGGACAGGAGAC 834 ASM-6 AGCCTGTTGAGTGGGCTGGGCCCAGCCGGCCCTTTTGATATGGTGTACTGGACAGGAGAC 834 ASM-7 AGCCTGTTGAGTGGGCTGGGCCCAGCCGGCCCTTTTGATATGGTGTACTGGACAGGAGAC 828 ************************************************************
ASM-1 ATCCCCGCACATGATGTCTGGCACCAGACTCGTCAGGACCAACTGCGGGCCCTGACCACC 900 ASM-5 ATCCCCGCACATGATGTCTGGCACCAGACTCGTCAGGACCAACTGCGGGCCCTGACCACC 894 ASM-6 ATCCCCGCACATGATGTCTGGCACCAGACTCGTCAGGACCAACTGCGGGCCCTGACCACC 894 ASM-7 ATCCCCGCACATGATGTCTGGCACCAGACTCGTCAGGACCAACTGCGGGCCCTGACCACC 888 ************************************************************
ASM-1 GTCACAGCACTTGTGAGGAAGTTCCTGGGGCCAGTGCCAGTGTACCCTGCTGTGGGTAAC 960 ASM-5 GTCACAGCACTTGTGAGGAAGTTCCTGGGGCCAGTGCCAGTGTACCCTGCTGTGGGTAAC 954 ASM-6 GTCACAGCACTTGTGAGGAAGTTCCTGGGGCCAGTGCCAGTGTACCCTGCTGTGGGTAAC 954 ASM-7 GTCACAGCACTTGTGAGGAAGTTCCTGGGGCCAGTGCCAGTGTACCCTGCTGTGGGTAAC 948 ************************************************************
ASM-1 CATGAAAGCACACCTGTCAATAGCTTCCCTCCCCCCTTCATTGAGGGCAACCACTCCTCC 1020 ASM-5 CATGAAAGCACACCTGTCAATAGCTTCCCTCCCCCCTTCATTGAGGGCAACCACTCCTCC 1014 ASM-6 CATGAAAGCACACCTGTCAATAGCTTCCCTCCCCCCTTCATTGAGGGCAACCACTCCTCC 1014 ASM-7 CATGAAAGCACACCTGTCAATAGCTTCCCTCCCCCCTTCATTGAGGGCAACCACTCCTCC 1008 ************************************************************
ASM-1 CGCTGGCTCTATGAAGCGATGGCCAAGGCTTGGGAGCCCTGGCTGCCTGCCGAAGCCCTG 1080 ASM-5 CGCTGGCTCTATGAAGCGATGGCCAAGGCTTGGGAGCCCTGGCTGCCTGCCGAAGCCCTG 1074 ASM-6 CGCTGGCTCTATGAAGCGATGGCCAAGGCTTGGGAGCCCTGGCTGCCTGCCGAAGCCCTG 1074 ASM-7 CGCTGGCTCTATGAAGCGATGGCCAAGGCTTGGGAGCCCTGGCTGCCTGCCGAAGCCCTG 1068 ************************************************************
ASM-1 CGCACCCTCAG------AATTGGGGG 1100 ASM-5 CGCACCCTCAG------1085 ASM-6 CGCACCCTCAG------AATTGGGGG 1094 ASM-7 CGCACCCTCAGGTACTTATCGTCCGTGGAAACCCAGGAAGGGAAAAGAAAGAATTGGGGG 1128 ***********
ASM-1 GTTCTATGCTCTTTCCCCATACCCCGGTCTCCGCCTCATCTCTCTCAATATGAATTTTTG 1160 ASM-5 ------ASM-6 GTTCTATGCTCTTTCCCCATACCCCGGTCTCCGCCTCATCTCTCTCAATATGAATTTTTG 1154 ASM-7 GTTCTATGCTCTTTCCCCATACCCCGGTCTCCGCCTCATCTCTCTCAATATGAATTTTTG 1188
ASM-1 TTCCCGTGAGAACTTCTGGCTCTTGATCAACTCCACGGATCCCGCAGGACAGCTCCAGTG 1220 ASM-5 TTCCCGTGAGAACTTCTGGCTCTTGATCAACTCCACGGATCCCGCAGGACAGCTCCAGTG 1145 ASM-6 TTCCCGTGAGAACTTCTGGCTCTTGATCAACTCCACGGATCCCGCAGGACAGCTCCAGTG 1214 ASM-7 TTCCCG TGA GAACTTCTGGCTCTTGATCAACTCCACGGATCCCGCAGGACAGCTCCAGTG 1248 ************************************************************
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ASM-1 GCTGGTGGGGGAGCTTCAGGCTGCTGAGGATCGAGGAGACAAAGTGCATATAATTGGCCA 1280 ASM-5 GCTGGTGGGGGAGCTTCAGGCTGCTGAGGATCGAGGAGACAAAGTGCATATAATTGGCCA 1205 ASM-6 GCTGGTGGGGGAGCTTCAGGCTGCTGAGGATCGAGGAGACAAAGTGCATATAATTGGCCA 1274 ASM-7 GCTAGTGGGGGAGCTTCAGGCTGCTGAGGATCGAGGAGACAAAGTGCATATAATTGGCCA 1308 *** ********************************************************
ASM-1 CATTCCCCCAGGGCACTGTCTGAAGAGCTGGAGCTGGAATTATTACCGAATTGTAGCCAG 1340 ASM-5 CATTCCCCCAGGGCACTGTCTGAAGAGCTGGAGCTGGAATTATTACCGAATTGTAGCCAG 1265 ASM-6 CATTCCCCCAGGGCACTGTCTGAAGAGCTGGAGCTGGAATTATTACCGAATTGTAGCCAG 1334 ASM-7 CATTCCCCCAGGGCACTGTCTGAAGAGCTGGAGCTGGAATTATTACCGAATTGTAGCCAG 1368 ************************************************************
ASM-1 GTATGAGAACACCCTGGCTGCTCAGTTCTTTGGCCACACTCATGTGGATGAATTTGAGGT 1400 ASM-5 GTATGAGAACACCCTGGCTGCTCAGTTCTTTGGCCACACTCATGTGGATGAATTTGAGGT 1325 ASM-6 GTATGAGAACACCCTGGCTGCTCAGTTCTTTGGCCACACTCATGTGGATGAATTTGAGGT 1394 ASM-7 GTATGAGAACACCCTGGCTGCTCAGTTCTTTGGCCACACTCATGTGGATGAATTTGAGGT 1428 ************************************************************
ASM-1 CTTCTATGATGAAGAGACTCTGAGCCGGCCGCTGGCTGTAGCCTTCCTGGCACCCAGTGC 1460 ASM-5 CTTCTATGATGAAGAGACTCTGAGCCGGCCGCTGGCTGTAGCCTTCCTGGCACCCAGTGC 1385 ASM-6 CTTCTATGATGAAGAGACTCTGAGCCGGCCGCTGGCTGTAGCCTTCCTGGCACCCAGTGC 1454 ASM-7 CTTCTATGATGAAGAGACTCTGAGCCGGCCGCTGGCTGTAGCCTTCCTGGCACCCAGTGC 1488 ************************************************************
ASM-1 AACTACCTACATCGGCCTTAATCCTG------GTTACCGTGTGTAC 1500 ASM-5 AACTACCTACATCGGCCTTAATCCTG------GTTACCGTGTGTAC 1425 ASM-6 AACTACCTACATCGGCCTTAATCCTGTCAGCCCCACATCCTTGCAGGTTACCGTGTGTAC 1514 ASM-7 AACTACCTACATCGGCCTTAATCCTG------GTTACCGTGTGTAC 1528 ************************** **************
ASM-1 CAAATAGATGGAAACTACTCCGGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC 1560 ASM-5 CAAA TAG ATGGAAACTACTCCAGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC 1485 ASM-6 CAAATAGATGGAAACTACTCCAGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC 1574 ASM-7 CAAATAGATGGAAACTACTCCGGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC 1588 ********************* **************************************
ASM-1 CTGAATCTGACCCAGGCAAACATACCGGGAGCCATACCGCACTGGCAGCTTCTCTACAGG 1620 ASM-5 CTGAATCTGACCCAGGCAAACATACCGGGAGCCATACCGCACTGGCAGCTTCTCTACAGG 1545 ASM-6 CTGAATCTGACCCAGGCAAACATACCGGGAGCCATACCGCACTGGCAGCTTCTCTACAGG 1634 ASM-7 CTGAATCTGACCCAGGCAAACATACCGGGAGCCATACCGCACTGGCAGCTTCTCTACAGG 1648 ************************************************************
ASM-1 GCTCGAGAAACCTATGGGCTGCCCAACACACTGCCTACCGCCTGGCACAACCTGGTATAT 1680 ASM-5 GCTCGAGAAACCTATGGGCTGCCCAACACACTGCCTACCGCCTGGCACAACCTGGTATAT 1605 ASM-6 GCTCGAGAAACCTATGGGCTGCCCAACACACTGCCTACCGCCTGGCACAACCTGGTATAT 1694 ASM-7 GCTCGAGAAACCTATGGGCTGCCCAACACACTGCCTACCGCCTGGCACAACCTGGTATAT 1708 ************************************************************
ASM-1 CGCATGCGGGGCGACATGCAACTTTTCCAGACCTTCTGGTTTCTCTACCATAAGGGCCAC 1740 ASM-5 CGCATGCGGGGCGACATGCAACTTTTCCAGACCTTCTGGTTTCTCTACCATAAGGGCCAC 1665 ASM-6 CGCATGCGGGGCGACATGCAACTTTTCCAGACCTTCTGGTTTCTCTACCATAAGGGCCAC 1754 ASM-7 CGCATGCGGGGCGACATGCAACTTTTCCAGACCTTCTGGTTTCTCTACCATAAGGGCCAC 1768 ************************************************************
ASM-1 CCACCCTCGGAGCCCTGTGGCACGCCCTGCCGTCTGGCTACTCTTTGTGCCCAGCTCTCT 1800 ASM-5 CCACCCTCGGAGCCCTGTGGCACGCCCTGCCGTCTGGCTACTCTTTGTGCCCAGCTCTCT 1725 ASM-6 CCACCCTCGGAGCCCTGTGGCACGCCCTGCCGTCTGGCTACTCTTTGTGCCCAGCTCTCT 1814 ASM-7 CCACCCTCGGAGCCCTGTGGCACGCCCTGCCGTCTGGCTACTCTTTGTGCCCAGCTCTCT 1828 ************************************************************
ASM-1 GCCCGTGCTGACAGCCCTGCTCTGTGCCGCCACCTGATGCCAGATGGGAGCCTCCCAGAG 1860 ASM-5 GCCCGTGCTGACAGCCCTGCTCTGTGCCGCCACCTGATGCCAGATGGGAGCCTCCCAGAG 1785 ASM-6 GCCCGTGCTGACAGCCCTGCTCTGTGCCGCCACCTGATGCCAGATGGGAGCCTCCCAGAG 1874 ASM-7 GCCCGTGCTGACAGCCCTGCTCTGTGCCGCCACCTGATGCCAGATGGGAGCCTCCCAGAG 1888 ************************************************************
ASM-1 GCCCAGAGCCTGTGGCCAAGGCCACTGTTTTGC TAG 1896 ASM-5 GCCCAGAGCCTGTGGCCAAGGCCACTGTTTTGC TAG 1821 ASM-6 GCCCAGAGCCTGTGGCCAAGGCCACTGTTTTGCTAG 1910 ASM-7 GCCCAGAGCCTGTGGCCAAGGCCACTGTTTTGCTAG 1924 ************************************ Fig. 4.17 Coding sequences of full-length ASM and new alternatively-spliced transcripts differ locally. Since all identified transcripts show sequence identity with full-length ASM except for described local differences, these transcripts are results of alternative splicing events. Grey parts indicate specific sequences differing from full-length ASM coding sequence. Stop codons in bold. Alignment was done using ClustalW.
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4.2.1 Structure of new ASM splice variants
One of the identified transcripts lacked the first 69 bp of exon 3, which resulted in a transcript length of 1.818 bp. The second transcript was characterized by a 20 bp intronic insertion derived from the end of intron 5 adjacent to exon 6 (exon 5a), leading to a transcript length of 1.907 bp. Two transcripts stood out due to a 40 bp intronic sequence derived from the beginning of intron 2 adjacent to exon 2 (exon 2a), which resulted in a 1.921 bp transcript (Fig. 4.18). Since Schuchman and colleagues (Schuchman et al. , 1991b) identified two ASM splice variants, termed ASM-2 and -3, and an additional splice variant, termed ASM-4, was deposited in the NCBI database GenBank by Reichwald et al. , the new transcripts were termed ASM-5, ASM-6 and ASM-7. Of note, the 40 bp intronic sequence from intron 2 found in ASM-7 is the same occurring in transcript ASM-2 and the 20 bp intronic sequence from intron 5 in ASM-6 is the same used in transcript ASM-4. However, the newly-identified splice variants were not identical to these transcripts (Fig. 4.18; Fig. 2.2). Both ASM-5 and ASM-6 were identified in human neuroglioma cells, whereas ASM-7 was found in human PBMCs. In total, 24% of the analysed ASM transcripts were alternatively-spliced.
Fig. 4.18 Schematic structure of new splice variants ASM-5, -6 and -7. Model of genomic alignment. Compared to ASM-1, in ASM-5 a part of exon 3 is excluded, which results in a 1.818 bp coding sequence. ASM-6 and ASM-7 display different intronic insertions, which generate transcript lengths of 1.907 bp and 1.921 bp, respectively, and create frameshifts including premature stop codons (indicated by asterisk) after 1.521 bp and 1.197 bp, respectively. In the depicted models of ASM exon-intron structure, the line represents the genomic sequence while black boxes stand for exons translated in ASM-1, grey boxes for exonic sequence translated into different amino acids due to a frameshift or sequence corresponding to introns in ASM-1, striped box for lacking exonic sequence and white boxes for exonic sequence following a premature termination codon. Scheme is drawn to scale.
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4.2.2 New splicing motifs are conserved in primates
We performed an in silico analysis concerning orthologous conservation of new ASM splice variants. Since the coding sequence of full-length ASM-1 is highly conserved among species, the comparison of ASM splice variants with orthologous transcripts is also reasonable. Newly-identified transcript ASM-6 shows exactly the same splicing patterns as the orthologous ASM transcripts identified in Pan troglodytes (GenBank Accession Number XM_001164317.1) and Macaca mulatta (Gen-Bank Accession Number XM_001110020.1). Newly-identified transcript ASM-7 harbours splicing patterns identical to orthologous ASM transcript expressed in Pongo abelii (GenBank Accession Number NM_001132129.1) (Fig. 4.19). Therefore, the utilization of splice sites at intronic sequences 2a and 5a which are used within ASM-6 and -7 is highly conserved. No orthologous transcripts were found for the newly-identified splice site in exon 3 used in ASM-5.
Fig. 4.19 Splicing patterns of novel transcripts ASM-6 and -7 are conserved in primates. GenBank search revealed orthologous ASM transcripts which show exactly the same splicing patterns as novel transcripts ASM-6 and -7. In the depicted models of ASM exon-intron structure, the line represents the genomic sequence while black boxes stand for exons translated in ASM-1, grey boxes for exonic sequence translated into different amino acids due to a frameshift or sequence corresponding to introns in ASM-1 and white boxes for exonic sequence following a premature termination codon. Scheme is drawn to scale.
4.2.3 Biochemical characterization of new ASM splice variants
On the predicted protein level, novel ASM splice variants display specific features in comparison with ASM-1 (631 amino acids, calculated molecular weight of 70 kDa). The protein derived from the new transcript ASM-5 covers 606 amino acids with a calculated molecular weight of 67 kDa. The catalytic domain is disrupted, since amino acids 362 to 384 are missing (Fig. 4.20). The protein derived from the new transcript ASM-6 is composed of 506 amino acids with a calculated molecular weight of 56 kDa, since the intronic insertion
74 4. Results creates a frameshift and subsequently introduces a premature stop codon TAG resulting in a truncated reading frame with 1.521 bp. Therefore, ASM-6 lacks 136 amino acids at its carboxyl terminus and carries a unique carboxyl-terminal peptide of 13 amino acids (VSPTSLQVTVCTK) instead, displaying only a fragmentary carboxy-terminal domain (Fig. 4.20 A). The protein derived from the new transcript ASM-7 consists of 398 amino acids and has a calculated molecular weight of 45 kDa, since a frameshift introduced by the intronic insertion creates a premature stop codon TGA, resulting in an open reading frame of 1.197 bp. Thus, ASM-7 lacks 267 amino acids at its carboxyl terminus and carries a unique carboxyl-terminal peptide of 38 amino acids (YLSSVETQEGKRKNWGVLC SFPIPRSPPHLSQYEFLFP) (Fig. 4.20 A). ASM-7 hence displays only a fragmentary catalytic domain, whereas the carboxy-terminal domain is completely missing. Taken together, alternative splicing events generating new variants ASM-5 to -7 affect the catalytic domain and the carboxyl-terminal domain.
A
ASM-1 MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALALALSDSRVLWAPAE 60 ASM-5 MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALA--LSDSRVLWAPAE 58 ASM-6 MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALA--LSDSRVLWAPAE 58 ASM-7 MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMG--LALALALALA--LSDSRVLWAPAE 56 ********************************** ********** ************
ASM-1 AHPLSPQGHPARLHRIVPRLRDVFGWGNLTCPICKGLFTAINLGLKKEPNVARVGSVAIK 120 ASM-5 AHPLSPQGHPARLHRIVPRLRDVFGWGNLTCPICKGLFTAINLGLKKEPNVARVGSVAIK 118 ASM-6 AHPLSPQGHPARLHRIVPRLRDVFGWGNLTCPICKGLFTAINLGLKKEPNVARVGSVAIK 118 ASM-7 AHPLSPQGHPARLHRIVPRLRDVFGWGNLTCPICKGLFTAINLGLKKEPNVARVGSVAIK 116 ************************************************************
ASM-1 LCNLLKIAPPAVCQSIVHLFEDDMVEVWRRSVLSPSEACGLLLGSTCGHWDIFSSWNISL 180 ASM-5 LCNLLKIAPPAVCQSIVHLFEDDMVEVWRRSVLSPSEACGLLLGSTCGHWDIFSSWNISL 178 ASM-6 LCNLLKIAPPAVCQSIVHLFEDDMVEVWRRSVLSPSEACGLLLGSTCGHWDIFSSWNISL 178 ASM-7 LCNLLKIAPPAVCQSIVHLFEDDMVEVWRRSVLSPSEACGLLLGSTCGHWDIFSSWNISL 176 ************************************************************
ASM-1 PTVPKPPPKPPSPPAPGAPVSRILFLTDLHWDHDYLEGTDPDCADPLCCRRGSGLPPASR 240 ASM-5 PTVPKPPPKPPSPPAPGAPVSRILFLTDLHWDHDYLEGTDPDCADPLCCRRGSGLPPASR 238 ASM-6 PTVPKPPPKPPSPPAPGAPVSRILFLTDLHWDHDYLEGTDPDCADPLCCRRGSGLPPASR 238 ASM-7 PTVPKPPPKPPSPPAPGAPVSRILFLTDLHWDHDYLEGTDPDCADPLCCRRGSGLPPASR 236 ************************************************************
ASM-1 PGAGYWGEYSKCDLPLRTLESLLSGLGPAGPFDMVYWTGDIPAHDVWHQTRQDQLRALTT 300 ASM-5 PGAGYWGEYSKCDLPLRTLESLLSGLGPAGPFDMVYWTGDIPAHDVWHQTRQDQLRALTT 298 ASM-6 PGAGYWGEYSKCDLPLRTLESLLSGLGPAGPFDMVYWTGDIPAHDVWHQTRQDQLRALTT 298 ASM-7 PGAGYWGEYSKCDLPLRTLESLLSGLGPAGPFDMVYWTGDIPAHDVWHQTRQDQLRALTT 296 ************************************************************
ASM-1 VTALVRKFLGPVPVYPAVGNHESTPVNSFPPPFIEGNHSSRWLYEAMAKAWEPWLPAEAL 360 ASM-5 VTALVRKFLGPVPVYPAVGNHESTPVNSFPPPFIEGNHSSRWLYEAMAKAWEPWLPAEAL 358 ASM-6 VTALVRKFLGPVPVYPAVGNHESTPVNSFPPPFIEGNHSSRWLYEAMAKAWEPWLPAEAL 358 ASM-7 VTALVRKFLGPVPVYPAVGNHESTPVNSFPPPFIEGNHSSRWLYEAMAKAWEPWLPAEAL 356 ************************************************************
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ASM-1 RTLRIGGFYALSPYPGLRLISLNMNFCSRENFWLLINSTDPAGQLQWLVGELQAAEDRGD 420 ASM-5 RTL------SSRENFWLLINSTDPAGQLQWLVGELQAAEDRGD 395 ASM-6 RTLRIGGFYALSPYPGLRLISLNMNFCSRENFWLLINSTDPAGQLQWLVGELQAAEDRGD 418 ASM-7 RTLRYLSSVETQEGKRKNWGVLCSFPIPRSPPHLSQYEFLFP------398 ***
ASM-1 KVHIIGHIPPGHCLKSWSWNYYRIVARYENTLAAQFFGHTHVDEFEVFYDEETLSRPLAV 480 ASM-5 KVHIIGHIPPGHCLKSWSWNYYRIVARYENTLAAQFFGHTHVDEFEVFYDEETLSRPLAV 455 ASM-6 KVHIIGHIPPGHCLKSWSWNYYRIVARYENTLAAQFFGHTHVDEFEVFYDEETLSRPLAV 478 ASM-7 ------
ASM-1 AFLAPSATTYIGLNPGYRVYQIDGNYSGSSHVVLDHETYILNLTQANIPGAIPHWQLLYR 540 ASM-5 AFLAPSATTYIGLNPGYRVYQIDGNYSRSSHVVLDHETYILNLTQANIPGAIPHWQLLYR 515 ASM-6 AFLAPSATTYIGLNPVSPTSLQVTVCTK------506 ASM-7 ------
ASM-1 ARETYGLPNTLPTAWHNLVYRMRGDMQLFQTFWFLYHKGHPPSEPCGTPCRLATLCAQLS 600 ASM-5 ARETYGLPNTLPTAWHNLVYRMRGDMQLFQTFWFLYHKGHPPSEPCGTPCRLATLCAQLS 575 ASM-6 ------ASM-7 ------
ASM-1 ARADSPALCRHLMPDGSLPEAQSLWPRPLFC 631 ASM-5 ARADSPALCRHLMPDGSLPEAQSLWPRPLFC 606 ASM-6 ------ASM-7 ------
B
Fig. 4.20 Novel variants ASM-5 to -7 differ regarding their putative protein sequence and protein domain structure. In comparison with ASM-1, ASM-5 lacks part of the catalytic domain, ASM-6 part of the carboxyl-terminal domain and ASM-7 part of the catalytic and the whole carboxy-terminal domain. ASM-6 and -7 carry unique carboxy-terminal peptides, indicated in grey. A. Putative protein sequences. Different protein domains are coloured: yellow - signal peptide (aa 1-48), light blue - saposin-B-domain (aa 91-167), purple - proline-rich- domain (aa 168-200), red - catalytic domain (aa 201-463), green - carboxy-terminal domain (aa 464-631). Mannose-6-phosphate sites (Asn 88, 177, 337, 397, 505, 522) are indicated in light green. Alignment was done using ClustalW. B. Schematic model of putative new ASM proteins. Mannose-6-phosphate sites are indicated by symbols. Scheme is drawn to scale.
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4.2.3.1 New ASM splice variants are expressed in a cell culture model
In order to analyse protein properties of the identified new ASM isoforms, the corresponding cDNAs were cloned into the expression vector FLAG-N2. Because of the putative N-terminal signal peptide, we expressed the ASM variants as C-terminal-tagged fusion proteins. HeLa cells were transiently transfected with ASM-FLAG constructs and harvested 48 h later. Western blot analysis showed that ASM-1 and each of the new splice variants ASM-5 to -7 were translated into protein and displayed the predicted sizes (Fig. 4.21).
Fig. 4.21 Expression of ASM-FLAG constructs in HeLa cells. Lysates were analysed by Western blotting 48 h after transfection. ASM-1 and each of the new isoforms ASM-5 to -7 displayed the predicted sizes. Expression of ASM-FLAG variants was detected using FLAG antibody. Lysates of vector-transfected cells served as negative control, GAPDH was detected for loading control. M indicates size marker.
4.2.3.2 New ASM splice variants are catalytically inactive in vitro
To determine the catalytic activity of the new ASM isoforms, H4 cells were transiently transfected with ASM-FLAG constructs. 48 h upon transfection cells were harvested and subjected to an in vitro biochemical enzyme-activity-assay (Gulbins and Kolesnick, 2000). ASM-1 over-expression selectively increased the ASM activity of cell lysates about 9-fold over endogenous levels (48.9 ± 1.3 nmol/mg/h, in comparison with control 5.4 ± 0.2 nmol/mg/h). In contrast to ASM-1, none of the alternatively-spliced variants exerted any
77 4. Results catalytic activity, despite well detectable expression of the proteins (ASM-5: 5.2 ± 1.1 nmol/mg/h; ASM-6: 5.2 ± 1.0 nmol/mg/h; ASM-7: 4.6 ± 1.6 nmol/mg/h; Fig. 4.22). To validate the impact of the FLAG-tag, we also expressed the variants without the C-terminal modification, which did not alter results (data not shown).
Fig. 4.22 Novel variants ASM -5 to -7 are catalytically inactive. Selectively ASM-1 over-expression increased ASM activity in comparison with cloning vector control. ASM- FLAG constructs were transiently expressed in H4 cells. Lysates were subjected to an in vitro enzyme-activity- assay 48 h upon transfection. Data indicate mean values of three independent experiments, error bars represent SD.
4.2.3.3 New ASM splice variants are catalytically inactive in vivo
Additionally, we investigated in vivo whether the new ASM isoforms modulate cellular ceramide and sphingomyelin levels. H4 cells were transiently transfected with ASM-FLAG constructs. A MALDI-TOF MS analysis showed that the ratio of ceramide to sphingomyelin 24 h upon ASM-1 over-expression was clearly elevated by 1.8-fold compared to control (p<0.01). In contrast, transfection of new ASM splice variants had the opposite effect and decreased ceramide to sphingomyelin ratio to between 26% and 38% of control (p<0.01 for ASM-6; p<0.05 for ASM-5 and -7) (Fig. 4.23). In total, we concluded that new ASM splice variants were catalytically inactive irrespective of whether they retained the catalytic domain or not and that they even exert an inhibitory effect on sphingomyelin breakdown.
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Fig. 4.23 New ASM splice variants modulate ceramide to sphingomyelin ratios. The ratio of ceramide (Cer) to sphingomyelin (SM) levels upon ASM-1 over-expression was significantly increased (p<0.01), upon ASM-5 to –7 over-expression significantly decreased (p<0.01 for ASM-6; p<0.05 for ASM-5 and -7) compared to cloning vector control. Lysates were subjected to MALDI-TOF MS for sphingolipid analysis 24 h after transfection of H4 cells. Data indicate mean values of three independent experiments, error bars represent SD. Statistical significance was calculated with respect to control (* p<0.05, ** p<0.01).
4.2.4 New ASM splice variants localize within different subcellular compartments
As described before, ASM wild type localizes in a dotted subcellular pattern and is located inside the lysosomes (see 4.1.2.2). To identify potential differences of ASM splice variants we investigated their subcellular localization. Due to the lack of specific antibodies we expressed splice variants as recombinant ASM-Cherry constructs with the fluorescent Cherry-tag on their C-termini. ASM-Cherry constructs were derived from ASM-FLAG constructs using infusion cloning. H4 cells were transiently transfected with ASM-Cherry constructs of ASM wild type and new ASM splice variants and fixed 48 h later. Cells were immunostained for the lysosomal marker protein LAMP1 and the endoplasmic reticulum marker protein PDIA4 to determine potential co-localization within lysosomes or endoplasmic reticulum, respectively. Subcellular localization patterns were investigated using fluorescence microscopy and co- localization with lysosomes or endoplasmic reticulum using confocal microscopy.
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As described above ASM wild type localized in a dotted subcellular pattern. Also ASM-7 was found to occur within dotted structures. In contrast, cells expressing ASM-5 and ASM-6 did not exhibit dotted structures (Fig. 4.24).
Fig. 4.24 Subcellular localization patterns of ASM-1 and new ASM splice variants. ASM-1 (A) and ASM-7 (D) localized in dotted subcellular patterns, whereas ASM-5 (B) and ASM-6 (C) did not. ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are representative cells investigated by fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three times.
Confocal microscopy analyses revealed that signals of ASM-Cherry wild type and ASM-7 constructs and signals of LAMP1 were co-localized (Fig. 4.25 A). In contrast, ASM-5 and ASM-6 constructs showed co-localization with endoplasmic reticulum marker PDIA4 (Fig. 4.25 B).
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Fig. 4.25 Co-localization of new ASM splice variants with subcellular organelles. ASM-1 and ASM-7 were localized within lysosomes, ASM-5 and ASM-6 within the endoplasmic reticulum. Confocal microscopy revealed co-localization of ASM-1 and ASM-7 with lysosomal marker protein LAMP1 (A) and of ASM-5 and ASM-6 with endoplasmic reticulum marker PDIA4 ( B). ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Marker proteins were detected by immunofluorescence with anti-LAMP1 and anti-PDIA4 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASM-Cherry constructs in red, marker proteins in green and merged signals. The indicated scale covers 10 µm. Co-localization experiments were reproduced three times.
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4.2.5 New ASM splice variants show differential expression patterns in human blood cells
In order to test if the newly-identified ASM transcripts are generally expressed, the cDNA of PBMCs from different donors was analysed by RT-PCR.
4.2.5.1 New ASM splice variants are detected specifically by RT-PCR
We designed distinct primer pairs for the exclusive amplification of each ASM transcript (Fig. 4.26 A), which were evaluated for target-specificity. PCR products were detected selectively if corresponding cDNA and primer pair were combined (Fig. 4.26 B). Each of the PCR products displayed the predicted size. Thus, designed oligonucleotides served for specific amplification of new ASM variants.
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Fig. 4.26 Designed oligonucleotides serve for specific amplification of new ASM splice variants. A. Distinct primer pairs were designed for specific amplification of each novel ASM splice variant. Exons are indicated by light grey boxes, transcript specific regions by symbol and dark grey boxes, primers by arrows. B. Primer specificity. Amplification of each novel ASM transcript using the respective primer pair resulted in a specific PCR-product of the expected size separated on a 1% agarose gel. Cloned transcripts were used as templates, water served as negative control. M refers to 100 bp size marker; indicated are DNA bands of 300 bp and 500 bp.
4.2.5.2 New ASM splice variants are differentially expressed in healthy volunteers
Using the established RT-PCR system, each of the newly-identified ASM splice variants could be detected in the cDNA from eight different donors. Notably, the expression level of each novel ASM splice variant varied in intra-individual comparison, and also the relative abundance of the splice variants differed between subjects (Fig. 4.27). PCR products were confirmed by sequencing (data not shown).
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Fig. 4.27 Expression patterns of ASM splice variants vary in human blood cells. cDNA of healthy volunteers (n=8) was analysed for the occurrence of each novel ASM transcript by RT-PCR and products were separated on a 1% agarose gel. ASM-1 and the new splice variants ASM-5, -6 and -7 exhibited both inter-individual as well as intra-individual differences in their expression. Water served as negative control. The second band of ASM-6 RT-PCR results from gDNA amplifications. M refers to 100 bp size marker; indicated are DNA bands of 300 bp and 500 bp.
4.2.5.3 New ASM splice variants are differentially expressed in depressed patients before and after antidepressant therapy
In order to investigate if expression patterns of new ASM splice variants were changed in depressed patients compared to healthy volunteers, we conducted additional RT-PCR experiments. We isolated RNA of whole blood derived from patients diagnosed for major depressive disorder at two different time points, before administering antidepressant drugs, i.e. in a complete untreated state, and after treatment for 28 days. The antidepressant therapy had been chosen according to the patients’ symptoms; therefore amitriptyline, mirtazapine and fluoxetine, respectively, were administered. The comparison of pre- and post treatment revealed an up-regulation of all ASM variants compared to reference gene HPRT, which was shown to be regulated independently from
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ASM (see thesis written by Jens Bode, 2010). Notably, strong effects were detected for the expression of ASM-5. Without antidepressant treatment, expression of ASM-5 was not detectable. In contrast, after administering of drugs ASM-5 was expressed to a high extent (Fig. 4.28). Compared to healthy volunteers (see 4.2.5.2) no obvious differences in expression patterns were observable. Depressed patients also displayed variations regarding their intra-individual expression patterns and in the relative abundance in inter-individual comparison.
Fig. 4.28 Expression patterns of ASM splice variants vary in blood cells of depressed patients before and after administering of antidepressants. cDNA of depressed patients was analysed for the occurrence of each novel ASM transcript by RT-PCR and products were separated on a 1% agarose gel. All ASM variants were up-regulated upon antidepressant therapy when compared to reference gene HPRT. New splice variant ASM-5 was not expressed without antidepressant treatment, but showed strong expression after administering drugs. Water served as negative control. Due to limited material obtained from patients control RT-PCR could not be repeated to avoid unspecific bands. M refers to 100 bp size marker; indicated are DNA bands of 300 bp and 500 bp.
85 4. Results
4.2.6 New splice variant ASM-5 exerts a dominant-negative effect on full- length Acid Sphingomyelinase
Based on expression differences of new splice variant ASM-5 detected in pre- and post- treatment conditions (see 4.2.5.3), we investigated functional effects of ASM-5.
4.2.6.1 Upon serum starvation ASM-5 localizes within lysosomes
As described above (see 4.2.4), ASM-5 localizes within the endoplasmic reticulum under basal conditions. Since cells of depressed patients are supposed to be in a stressed state, we conducted experiments under stress conditions, induced by serum starvation. For this purpose we transiently transfected H4 cells with ASM-5-Cherry construct. 48 h upon transfection cells were shifted to serum-free medium. 24 h later cells were fixed and immunostained for LAMP1 and PDIA4 to determine potential co-localization within lysosomes or endoplasmic reticulum, respectively. Subcellular localization patterns were investigated using fluorescence microscopy and co-localization with lysosomes or endoplasmic reticulum using confocal microscopy.
ASM-5 was found to occur within dotted structures under serum-starving conditions (Fig. 4.29), in contrast to basal conditions (data equate to fig. 4.24B).
86 4. Results
Fig. 4.29 Subcellular localization pattern of ASM-5 upon serum starvation. ASM-5 localized in a dotted subcellular pattern under serum-starving conditions, in sharp contrast to basal conditions (see 4.2.4). ASM-Cherry constructs were detected by fluorescence (Excitation: 543 nm, Emission: 560 – 615 nm). Shown are representative cells investigated by fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three times.
To evaluate co-localization of ASM-5 with marker proteins under starving conditions, confocal microscopy analyses were conducted. Signals of ASM-5-Cherry and signals of LAMP1 were co-localized, indicating that ASM-5 was localized inside the lysosomes (Fig. 4.30).
Fig. 4.30 ASM-5 localizes within lysosomes upon serum starvation. Confocal microscopy revealed co-localization of ASM-5-Cherry construct with lysosomal marker protein LAMP1. ASM-Cherry construct was detected by fluorescence (Excitation: 543 nm, Emission: 560 – 615 nm). LAMP1 was detected by immunofluorescence with anti-LAMP1 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASM-Cherry constructs in red, LAMP1 in green and merged signals. The indicated scale covers 10 µm. Co-localization experiments were reproduced three times.
87 4. Results
4.2.6.2 ASM-5 is a dominant-negative ASM isoform
Since ASM-1 and ASM-5 both show lysosomal localization under serum-starving conditions, interactive effects on ASM activity levels are possible. Therefore, we transiently co- transfected HeLa cells with ASM-1 and ASM-5-FLAG constructs under starving and basal conditions. Starving protocol was applied as described for localization studies in 4.2.6.1. Cell lysates were subjected to an in vitro biochemical enzyme-activity-assay. Exogenous catalytic activity of ASM-1 over-expression was set on 100% for each condition. Compared to the respective reference, ASM-5 did not exert catalytic activity (starving condition: 0.2% ± 1.3 of ASM-1 activity; basal condition: 1.3% ± 1.3 of ASM-1 activity). Upon serum starvation over-expression of a mixture of ASM-1 and ASM-5 lead to a significant reduction of ASM activity. Adding 25% of ASM-5 to the transfection mixture reduced ASM activity to 60.2% compared to over-expression of ASM-1 (SD ± 20.0; p<0.05), while the addition of 50% of ASM-5 reduced ASM activity to 52.5% of ASM-1 activity (SD ± 12.7; p<0.05). In contrast, no effect of varying amounts of ASM-5 over-expression on catalytic activity of ASM-1 was observed under basal conditions. Compared to ASM-1, over- expression of a mixture of ASM-1 and ASM-5 did not change ASM activity levels (102% ± 12.2 of ASM-1 activity and 97.8 ± 4.1 of ASM-1 activity) (Fig. 4.31). These results imply a dominant-negative effect of ASM-5 on ASM-1 selectively upon serum starvation.
88 4. Results
Fig. 4.31 ASM-5 exerts a dominant-negative effect on ASM-1 upon serum starvation. Under starving and basal conditions ASM-1 over-expression increased ASM activity, in contrast to ASM-5 and vector control over-expression. Over-expression of a mixture of ASM-1 and ASM-5 lead to a significant reduction of ASM activity under starving conditions, which was not observed under basal conditions. ASM- FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzyme- activity-assay 72 h upon transfection. Results are given in exogenous ASM activity levels in percentage relative to activity of full-length ASM-1 (100%). Displayed data represent mean values of three independent experiments, error bars represent SD. Statistical significance was calculated with respect to ASM-1 (* p<0.05).
89 5. Discussion
5. Discussion
In this study, we investigated ASM sequence variations on DNA and RNA levels. The aim was to gain insight into the molecular mechanisms of increased ASM activity levels which were detected within depressed patients. Regarding the DNA level we did not detect sequence variations which harboured the potential to increase the intrinsic activity of ASM enzymes under experimental conditions. Nevertheless, we identified differential characteristics of known common and rare ASM sequence variations which were detected within our patient collective. The data concerning catalytic activity and subcellular localization give further hints for the importance of proper processing for the regulation of ASM. Regarding the RNA level we were able to identify to date undescribed ASM splice variants and to characterize the biochemical properties of their corresponding proteins. We could show that alternatively-spliced ASM variants are able to exert regulative functions on cellular ASM levels and that their RNA expression is up-regulated by antidepressant drugs. In total, this study presents a further piece of the enigma of ASM regulation in health and disease.
5.1 DNA sequence variations impact Acid Sphingomyelinase activity
We investigated the impact of ASM DNA sequence variations resulting in missense mutations of the ASM protein sequence on enzymatic activity and subcellular localization patterns. Compared to ASM wild type, most of the ASM variants displayed specific characteristics. The opposite effect of NPD mutations which lack full enzymatic activity, namely an intrinsic increase in ASM activity, was not detected. To summarize results, increased ASM activity levels detected in depressed patients are probably not caused by the mere variation of ASM sequence. However, ASM sequence variations might result in ASM enzymes which could be prone to differential or dysfunctional processing or regulation.
90 5. Discussion
5.1.1 The C-terminal domain harbours essential parts for activation and secretion of Acid Sphingomyelinase
Several findings indicate that alterations in the C-terminal region impact catalytic activity and subcellular localization of ASM. The C-terminal domain of ASM was shown to be highly important for ASM activation and cellular trafficking, mostly due to several posttranslational modifications. So are the essential glycosylation sites which ensure proper lysosomal localization positioned at the C-terminus (Ferlinz et al. , 1997). Also, it is the disulfide bonds at the C-terminus, which are highly necessary for correct lysosomal localization and catalytic activity of ASM (Lee et al. , 2007). The C-terminal phosphorylation site p.S508 was reported to be essential for activation of ASM by protein kinase δ (Zeidan and Hannun, 2007a). The drastic amino acid substitution of serine by alanine at this position (p.S508A) has several implications for ASM activity and localization: On one side it alters a phosphorylation site and therefore the potential of being fully activated, on the other side it prevents its ability of being secreted (Jenkins et al. , 2010a). In this study, we investigated the effect of the common ASM variation p.G506R. Since this constitutes an amino acid exchange in such a close proximity to the important ASM phosphorylation site p.S508, it could exert influence on ASM properties. Concerning catalytic activity we detected a statistically significant difference of 25% with ASM variant p.G506R being less catalytically active than ASM wild type (Fig. 4.14). Possibly, the phosphorylation of serine 508 by protein kinase δ could be affected by the exchange of glycine for arginine on ASM position 506, which constitutes a dramatic alteration of protein sequence. This would mean an inability of ASM p.G506R to reach its full activity. Equivalently, the same trend was detected for ASM variation p.G506R and ASM wild type in combination with ASM variation p.A487V. Only a slight difference in catalytic activity was measured for ASM variation p.G506R and ASM wild type in combination with ASM variation p.S508A (Fig. 4.14). Since the essential phosphorylation site on position 508 is altered in both variants, no full catalytic activity is exerted, regardless of an exchange at position 506. In contrast, these two variants display differential cellular localization patterns. The ASM variant covering a combination of variations p.G506R and p.S508A displays very low protein expression in our Western blot analyses (Fig. 4.3) and is trapped inside the endoplasmic reticulum instead of trafficking to lysosomes (Fig. 4.16) – in contrast to ASM variants featuring only the singular variations, which show lysosomal localization (Fig. 4.7; 4.16). Two mechanisms could be responsible for this effect: ASM variant p.G506R/S508A may be rapidly degraded upon being trapped inside the ER or could
91 5. Discussion be secreted into extracellular space to a major part. To get insight into the cellular fate, it should be investigated if ASM variant p.G506R/S508A is detected inside the Golgi which serves as sorting point for the two ASM forms, the lysosomal one and the secreted one (Jenkins et al. , 2010a). In summary, these results imply a regulatory effect of the amino acid at position 506 and stress the importance of the C-terminal region around position 506/508 for trafficking and secretion of ASM. Further investigations should be conducted to get insight into the exact mechanisms leading to differential trafficking of ASM enzymes.
5.1.2 Processing of Acid Sphingomyelinase is essential for its regulation in pathologic context
Several studies about Niemann-Pick disease type A and B give insight into the relation between ASM genotype and corresponding ASM activity (Simonaro et al. , 2002; Sikora et al. , 2003). However, the residual ASM activity detected within patients did not always correlate with the severity of clinical symptoms, especially for NPD type B (Chan et al. , 1977; Sogawa et al. , 1978; Dawson and Dawson, 1982). Also, studies focussing on ASM variations differed regarding their results about catalytic activity and localization patterns, depending on the experimental system – using patients’ material or over-expression strategies of recombinant constructs. For example, ASM variant p.G242R exerted a catalytic activity of 15% wild type activity in a patient’s lymphoblasts, whereas the recombinant ASM variant which was over-expressed in COS-1 cells reached a level of about 40% wild type activity (Takahashi et al. , 1992b). Another example gives ASM variant delR608: Whereas Jones et al. (2008) detect this variant inside the lysosomes in patients’ fibroblasts, Jenkins et al. (2010b) find the recombinant construct expressed in HEK 293 cells trapped within the endoplasmic reticulum as did Lee et al. in COS-1 cells (2007). Therefore, over-expression studies are indeed useful for evaluating intrinsic properties of ASM variants, but are at the same time somewhat limited, since they do not resemble the processing and regulation of a pathologic cellular context. In our study, we detected the two rare ASM sequence variations p.P325A and p.A487V within a collective of patients suffering from depression. These variants were previously defined as Niemann-Pick type B mutations according to their reduced ASM activity levels in patients’ fibroblasts and blood cells (Simonaro et al. , 2002). This finding seems to constitute a sharp contrast to the assumed underlying genetic defects,
92 5. Discussion where depressed patients showed increased, patients suffering from NPD decreased ASM activity. Whereas ASM p.P325A was indeed catalytic inactive (Fig. 4.8) but was located inside the lysosomes (Fig. 4.10), ASM p.A487V displayed the same properties as did ASM wild type (Fig. 4.11 and 4.13). Obviously, ASM trafficking is of highest importance for ASM processing, since only proper lysosomal localization allows full catalytic activity in vivo (Ferlinz et al. , 1994; Ni, 2006). So in principle for both variants the possibility is provided to be processed and activated in vivo due to lysosomal localization under our experimental conditions. However, processing of p.P325A can probably not compensate the alteration within the catalytic domain. In contrast, variant p. A487V displays an intact catalytic domain and therefore could be processed and activated – probably depending on cellular context. As a third category, Niemann-Pick disease type A variant ASM p.M381I has an altered catalytic domain and is trapped inside the ER (Fig. 4.7), which prevents any further processing. Therefore, variants located inside the lysosome and featuring an intact catalytic domain could exert varying catalytic activities in dependence of their cellular context. In total, cellular processing of ASM is probably highly influenced by cell type and pathologic context, resulting in differential modifications of intrinsic properties in vivo . Thus, experimental conditions could account for restraints in detecting differences between ASM variants regarding differential regulation. Further studies should concentrate on cell specific ASM processing and regulation to get insights into disease related mechanisms.
5.2 Alternative splicing impacts Acid Sphingomyelinase activity
In our study we identified the three additional human alternatively-spliced ASM transcripts ASM-5, ASM-6 and ASM-7, which are catalytically inactive and distinctly expressed within individuals. Since alternatively-spliced variant ASM-5 exerts a dominant-negative effect on full-length ASM, cellular ASM activity levels could be regulated by alternative splicing. This mechanism seems to be induced by chronic administering of antidepressant drugs. ASM alternative splicing therefore could be a potential mechanism for ASM regulation. Investigating ASM alternative splicing especially within brain tissue and blood cells could offer insights into stress and disease related mechanisms.
93 5. Discussion
5.2.1 Alternative splicing of Acid Sphingomyelinase could predominate in brain and blood cells
In this study, we identified three new human alternatively-spliced ASM transcripts. In our approach, the cloning of PCR amplified full-length transcripts, alternatively-spliced ASM transcripts occurred to a higher extent than expected (Quintern et al. , 1989; Schuchman et al. , 1991b). From the analysis by Schuchman and colleagues, the rate of alternatively-spliced transcripts was estimated to be about 10% while in our hands, every fourth transcript arose from alternative splicing. The diverging splicing rates might be due to the different tissue under investigation. Splice variants ASM-2 and ASM-3 were identified within cDNA libraries of fibroblast, placental or testis tissue. However, alternative splicing is particularly important for neuronal functions (Grabowski and Black, 2001) and the brain exhibits the highest number of tissue-specific splice forms (Xu et al. , 2002). Two of our new ASM splice variants were isolated from a human neuroglioma cell line, and previously described ASM-4 was also derived from human brain tissue. The third new ASM splice variant was identified within human primary blood cells. This could be of interest, since the main features of mammalian stress response are changes within the peripheral and central nervous system, modification of blood cell composition, and alterations in many gene products. For example, alternative splicing of the acetylcholinesterase was reported as a stress response in brain and blood cells (Pick et al. , 2004). Similarly, alternative splicing of ASM may be particularly abundant in brain and blood cells as part of the cellular stress response.
5.2.2 Acid Sphingomyelinase displays a defined set of conserved splicing motifs
In general, the modification of protein properties by alternative splicing is an often- discovered phenomenon (Okazaki et al. , 2002; Zavolan et al. , 2003). In particular, concerning enzymes, alternative splicing is able to affect all enzymatic properties, such as
substrate affinity, substrate binding, V max , activity regulation and catalytic properties (Stamm et al. , 2005). However, alternative splicing of ASM generally results in catalytically inactive isoforms, as judged by biochemical in vitro assays. The only functional transcript encoding an active enzyme thus far is transcript ASM-1 encompassing exon 1 to 6 (Fig. 4.22). The
94 5. Discussion
ASM enzyme is composed of a putative signal peptide, a saposin-B-domain, a proline-rich domain, a calcineurine-like phosphoesterase domain and a carboxy-terminal domain. Compared to this, alternatively-spliced forms lack essential parts. A common motif is the differential splicing of exon 3, which leads to the generation of ASM isoforms with a disrupted catalytic domain (e.g. ASM-2, ASM-3). As a variation of this theme, we discovered a splice form in which exon 3 was only partly missing (ASM-5). A second common motif is the inclusion of 40 bp intronic sequence adjacent to the 3’ end of exon 2 (herein referred to as exon 2a) into the mature transcript (found in ASM-2, ASM-7). If not compensated by the out- splicing of exon 3 (as in ASM-2), the inclusion of exon 2a ultimately provokes a shift in the reading frame (Fig. 2.2, 4.18 and 4.20). Either way leads to the disruption of the catalytic domain. The last common motif is the inclusion of 20 bp intronic sequence adjacent to the 5’ end of exon 6 (herein referred to as exon 5a) into the mature transcript, which also provokes a frameshift and the generation of truncated proteins. The exon 5a is present in the transcripts ASM-4 and ASM-6. However, since a second alternative splicing event affecting exon 2 and thus the catalytic domain is encountered in ASM-4, ASM-6 is the only splice form retaining the complete catalytic domain except for functional ASM-1. Nevertheless, just like the other splice forms, ASM-6 was found to be inactive in our biochemical assays. In total, alternative splicing selectively affects the integrity of the catalytic and C-terminal domain. Interestingly, the ASM sequence variations which lead to catalytically-inactive enzymes and cause Niemann-Pick disease type A and B, are also selectively located within the ASM sequence coding for the catalytic domain (75%) or the C-terminal part (25%) (Ferlinz et al. , 1991; Levran et al. , 1991; Levran et al. , 1992; Takahashi et al. , 1992a; Takahashi et al. , 1992b; Levran et al. , 1993; Ferlinz et al. , 1995; Simonaro et al. , 2002; Rodriguez-Pascau et al. , 2009). Therefore, changes solely within the C-terminal part can also abolish enzymatic activity of ASM. Of note, the usage of the motifs described above is conserved among different species. GenBank search revealed orthologous ASM splice forms identical to variants ASM-2, -6 and -7 expressed in different primates. Therefore, ASM splicing covers a defined set of motifs which are evolutionary conserved.
95 5. Discussion
5.2.3 Acid Sphingomyelinase activity is regulated by alternative splicing
Since ASM plays an essential role in the fragile balance of the rheostat, alternative splicing occurs within a highly-regulated context. ASM enzymatic activity exerts strong consequences for cellular functioning, deciding between apoptosis and proliferation, and is increased upon stress signalling (Carpinteiro et al. , 2008). External stimuli like cellular stress and receptor signalling as well are an important source of changes in cellular processes and also affect splice site selection to a great extent. The switch from enzymatic activity to inactivity by alternative splicing, triggered by stress signals, is a well-known phenomenon (Kaufer et al. , 1998). According to this, dysbalanced ASM activity upon occurrence of stress stimuli could be rebalanced by alternative splicing events. Under basal conditions, the rheostat is balanced, with ASM exerting basal enzymatic activity. Under stress conditions, ASM is fully activated, exerting increased enzymatic activity, which results in an accumulation of ceramide. The dysbalanced rheostat could be rebalanced by a switch in splice site selection via the spliceosome, leading to an increase in inactive ASM enzymes, which would counter the effects of the dysbalanced rheostat. Experimental evidence for this assumption is offered by our in vivo results. Ratios of cellular ceramide to sphingomyelin contents upon new ASM splice variants expression are significantly lower than those of the control condition (Fig. 4.23) and equate to untransfected cells (data not shown). Since transfection of cells always means cellular stress, the control condition is also subjected to elevated endogenous ASM activity levels. Expression of catalytic inactive ASM splice variants clearly decreases the ceramide to sphingomyelin ratio, modulating the stress-induced increase in ASM activity. The result is a normalized cellular ceramide to sphingomyelin ratio, which corresponds to the ratio within cells in an unstressed state. The mechanisms of how primarily inactive ASM isoforms could contribute to total cellular ASM activity probably depend on the different structural and functional categories of ASM splicing. It was reported that activated ASM forms dimers in particular via the most carboxy- terminal located cysteine (Qiu et al. , 2003). Thus, inactive ASM isoforms which retain the C- terminus could act as dominant-negative modulators of ASM activity by binding to active ASM isoforms (Stasiv et al. , 2001; Zhang et al. , 2010). This mechanism could take effect on ASM-5, which was shown to inhibit ASM activity upon co-expression with full-length ASM and serum starvation (Fig. 4.31). Since ASM-5 displays an intact C-terminus, it should be capable of dimerizing – despite its disrupted catalytic domain. As shown in figure 4.30, ASM-5 translocates into the lysosome upon serum starvation, where it could interact with
96 5. Discussion full-length ASM, leading to a dominant-negative effect on active ASM in this stress condition. For ASM-6 and -7, different mechanisms should take effect due to their truncation. Splicing events which result in truncated variants are often subjected to nonsense-mediated decay (Stamm et al. , 2005). Three-quarters of alternatively-spliced transcripts featuring a frameshift leading to a premature stop codon are supposed to undergo nonsense-mediated decay and are switched off, probably because of their instability (Lewis et al. , 2003; Wollerton et al. , 2004). Therefore, the cellular ASM activity level could be decreased by switching off ASM transcripts -6 and -7 after a short life within the cell. Additional hints for a regulative role of alternative spliced isoforms are given by our results about expression patterns of new ASM splice variants within individuals. Full-length ASM-1 and each of the new ASM splice variants were expressed to a different extent upon inter- individual comparison. Also, regarding the intra-individual level, highly-differential expression patterns of ASM splice variants were observed (Fig. 4.27). These findings clearly show that alternative splicing of ASM is regulated and therefore bears relevance for cellular processes. Expression patterns of patients suffering from depression show that ASM splice variants are differentially regulated by antidepressant drugs. Despite the small case number and the complexity of influencing factors it is obvious that especially ASM-5 is dramatically up-regulated after chronic administering of different antidepressants (Fig. 4.28). Effects of antidepressants on the selective up-regulation of a special splice variant were already shown for further stress- and depression related molecules (D'Sa et al. , 2005; Funato et al. , 2006). Antidepressant drugs therefore could be able to inhibit ASM activity by two different mechanisms: Functional inhibitors like desipramine and imipramine inhibit ASM activity via the detachment of ASM from the lysosomal membrane and subsequent proteolytic cleavage inside the lysosome (Kölzer et al. , 2004). Since all ASM transcripts showed increased expression levels upon administering of antidepressants, this degradation in turn could lead to enhanced ASM transcription to ensure supply. Potent ASM inhibitors like fluoxetine and amitriptyline could also inhibit ASM activity via the induction of the dominant-negative splice variant ASM-5. Accordingly, the effect exerted by ASM-5 due to its unique properties probably is of strong importance for cellular ASM regulation. Further studies should elucidate the exact relation between splicing patterns and respective cellular ASM activities. In total, elevated cellular ASM activity could be regulated by splicing events, generating inactive variants for acting as modulators of wild type ASM. In pathologic context, this mechanism could be initiated by the effect of antidepressants.
97 6. Bibliography
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7. Appendix
7.1 Chemicals Agarose Biozym, Oldendorf APS Merck, Darmstadt Ampicillin Sigma-Aldrich, Munich β-Mercaptoethanol Roth, Karlsruhe Bacto-Agar Roth, Karlsruhe Bromphenol blue Merck, Darmstadt Calciumchloride (CaCl 2) Merck, Darmstadt Chloroform Roth, Karlsruhe Desoxyribonucleictriphosphat-Set (dNTP) Peqlab, Erlangen D, L-Dithiothreitol (DTT) Amersham, Freiburg Dimethylsulfoxide (DMSO) Roth, Karlsruhe DMEM-Medium Biochrom, Berlin ECL-Solution Amersham, Freiburg Ethanol Merck, Darmstadt Ethidiumbromide Roth, Karlsruhe FCS Biochrom, Berlin Glacial acetic acid Merck, Darmstadt GFP Amaxa, Cologne L-Glutamine Roth, Karlsruhe Glycin Merck, Darmstadt Isopropanol Roth, Karlsruhe Yeast extract Roth, Karlsruhe Kaliumchloride (KCl) Sigma-Aldrich, Munich Kaliumdihydrogenphosphate (KH 2HPO 4) Merck, Darmstadt Kanamycin Sigma-Aldrich, Munich Magnesiumchloride (MgCl 2) Sigma-Aldrich, Munich Magnesiumsulfate (MgSO 4) Merck, Darmstadt Milk powder Roth, Karlsruhe Natriumchloride (NaCl) Roth, Karlsruhe Natriumdodecylsulfate (SDS) Amersham, Freiburg Natriumhydrogenphosphate (Na 2HPO 4) Sigma-Aldrich, Munich Nonidet P40 Sigma-Aldrich, Munich Oligonucleotides Operon, Huntsville Protease-Inhibitor Mix (CompleteMini ) Roche, Mannheim Qiazol Qiagen, Hilden Sphingomyelin 14 C Perkin Elmer, Waltham, USA TEMED Sigma-Aldrich, Munich Tris(hydroxymethyl)-aminomethane (Tris) Roth, Karlsruhe Triton-X-100 AppliChem, Darmstadt Trypsin Biochrom, Berlin Trypton Roth, Karlsruhe Tween20 Roth, Karlsruhe
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7.2 Technical device
Biofuge Centrifuge pico Heraeus, Hanau Block heater Grant Boekel, UK Casy cell counter Innovatis, Reutlingen Cooling centrifuge Eppendorf, Hamburg Experion System BioRad, Munich Fluoreszenzmicroscope Axiovert 135 Zeiss, Jena Freezer Liebherr Bulle, Switzerland Gel system for western blotting BioRad, Munich Hicclave HV-85 Autoklav Schubert-Weiss, Munich Ice machine Scotsman, Herborn Incubator Hera cell Heraeus, Hanau Laser scanning microscope DM IRE2 Leica, Solms Light microscope DM IL HC Bio Leica, Solms Microplate Reader BioRad, Munich Nitrogen tank air liquide, Düsseldorf Oven Memmert, Schwabach pH-Meter WTW, Weilheim Photometer Eppendorf, Hamburg Pipettes Eppendorf, Hamburg/ Gilson, Frankreich VWR, Darmstadt Pipetboy Schubert&Weiss, Munich Power Supply Electrophoresis EPS 301 Amersham, Freiburg QuantityOne Fluor-S ™ MultiImager TKA-Lab, Niederelbert Sterile hood Heraeus, Hanau Thermomixer comfort Eppendorf, Hamburg Thermocycler Sensoquest, Göttingen Typhoon Amersham, Freiburg Vortex Mixer NeoLab, Heidelberg
7.3 Auxiliary material
Cell culture flasks TPP, Switzerland Cell culture plates TPP, Switzerland Eppendorf tubes Eppendorf, Hamburg Medical gloves Kimberly-Clark, UK Parafilm Pechiney, Chicago, USA Pasteurpipettes Roth, Karlsruhe Pipette Tips Eppendorf, Hamburg PVDF Membrane Millipore, Schwabach Serologic Pipettes GreinerBio-One, Frickenhausen Tissues Roth, Karlsruhe
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