Inhalt

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   Summary
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   Zusammenfassung
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  Introduction
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   Lysosomes and the endolysosomal system
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   Uptake of compounds into the lysosomes
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   The lysosomal proteom
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   Synthesis of lysosomal proteins
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   Targeting of proteins to the endolysosomal system
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   Maturation of lysosomal hydrolases
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   The Sulfatase enzyme family
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   Generation of formylglycine
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   Catalytic mechanism of type I sulfatases
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   Mammalian sulfatases
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   Arylsulfatase G
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   Glycosaminoglycans
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   Biosynthesis of heparan sulfate and heparin
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   Degradation of heparan sulfate and heparin
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  Lysosomal storage disorders
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   Mucopolysaccharidoses
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   Therapeutic options in mucopolysaccharidoses
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   Aims of the study
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  Materials
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   Laboratory equipment
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   Laboratory materials
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   Chemicals and reagents
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   Monosaccharides
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   Kits
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  Antibodies
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   Primary antibodies
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   Secondary antibodies
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   Radioactive Compounds
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   Antibiotics
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   Peptides
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   Thyroid hormones and hormone sulfates
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   Enzymes
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  Oligonucleotide primers
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   Genotyping primer for Arsg KO mice
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   Primer for 3' homologous recombination of Arsg KO mice
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   Standard sequencing primers
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   Primers for generation of untagged human ARSG
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   Mutagenesis primers for preparations of human ARSG mutants
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   Primers for amplification of human ARSG cDNA
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   Internal sequencing primers for human ARSG
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   Primers for human ARSG exon amplification
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   Cloning primer for the murine ARSG
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   Mutagenesis primers for preparations of murine ARSG mutants
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   Primers for the generation of N-terminal c-Myc-tagged murine arylsulfatase G
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   Sequencing primers of murine ARSG
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   Common buffer and solutions
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   Protein and DNA markers
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   Plasmids
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   Bacterial strains
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   Media for cultivation of Escherichia coli
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  Mammalian cell lines
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   Mouse embryonic fibroblasts
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   Established cell lines
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   Putative patients
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   Cell culture media
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   Mouse strains
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   Software & Onlinetools
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  Methods
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  Molecular biology
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   Purification of plasmid DNA
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   Restriction digest
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   Dephosphorylation of DNA
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   Agarose gel electrophoresis
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   DNA extraction from agarose gels and PCR purification
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   Ligation of DNA
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   Working with RNA
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   DNA sequencing
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   TA cloning
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   Extraction of genomic DNA from cells and tissues
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   Polymerase chain reaction
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   Cloning of murine arylsulfatase G
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   Add-around PCR
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   Molecular biological analysis of potential MPS IIIE patients
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  Cultivation of bacteria
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   Preparation of chemocompetent Escherichia coli
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   Transformation of chemocompetent Escherichia coli
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   Blue-white screen
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   Storage of Escherichia coli strains
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  Cell culture methods
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   General cell culture conditions
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   Cryoconservation and revitalisation of mammalian cells
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   Transfection of mammalian cells
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   Generation of stably transfected, mammalian cell lines
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   Transient transfections of mammalian cells
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   Immortalisation of mouse embryonic fibroblasts
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   Generation of cell pellets and harvesting of conditioned medium
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   Protein mass production in mammalian cells
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   Methotrexate induced gene amplification in chinese hamster ovary DG44 cell
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   Dialysis of fetal calf serum
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   Preparation and cultivation of primary BM-derived macrophages
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  Biochemical methods
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   Preparation of cell and tissue homogenates
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   Protein concentration determination
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   Purification of human RGSHis-tagged arylsulfatase G
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   Storage of purified human arylsulfatase G
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   Activity assays for lysosomal standard enzymes
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   SDS-PAGE
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   Non-reducing SDS-PAGE
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   Blue silver staining of polyacrylamide gels
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   Silver staining of polyacrylamide gels
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   Drying of SDS polyacrylamide gels
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   Semi-dry Western blot
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   Identification of FGly-containing peptides of hARSG by MALDI-ToF MS
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   Labelling of formylglycine with Alexa Fluor 488 hydroxylamine
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   Trichloroacetic acid precipitation of proteins
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   Ammonium sulfate precipitation of proteins
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   Cleaning of Pansorbin
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   Immunoprecipitation of radiolabelled arylsulfatase G
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   Immunoprecipitation of endogenous arylsulfatase G for activity assays.
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   Size exclusion chromatography for determining molecular weights
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   Subcellular fractionation of mouse livers and tritosomal purification
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   PERCOLL gradient for purification of lysosomes from mouse liver
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   Sulfatase activity assays with sulfatase pseudosubstrates
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   Activity assays with monosaccharide substrates
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   Sulfate release detection with barium ions
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   Desulfation assays with thyroid hormone sulfates
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  Glycobiological methods
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   Enzymatic deglycosylation of glycoproteins
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   Glycosaminoglycan extraction from mouse tissues for quantification
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   Glycosaminoglycan extraction from mouse tissues for MS analysis
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   Glycan Reductive Isotope Labeling-LC/MS
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   Labelling of monosaccharides with 2-aminoacridone
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   Acylation of N-unsubstituted glucosamines
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   Labelling of monosaccharides with anthranilic acid
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   Acetylation of D-glucosamine 3-O-sulfate
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  Cell biological methods
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   Metabolic labelling of proteins with radiolabelled methionine and cysteine
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   In vivo inhibition of proteases
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   Inhibition of glucosidases by 1-deoxynojirimycin
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   Disruption of lysosomal targeting and processing by ammonium chloride
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   Coating of cover slips with poly-L-lysine
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   Immunofluorescence of mammalian cells with para-formaldehyde fixation
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   Staining of cell nuclei by 4',6-Diamidino-2-phenylindole
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  Histological methods
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   Mouse breeding
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   Mouse transfer
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   Passive perfusion with para-formaldehyde
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   Active perfusion with para-formaldehyde
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   Generation of semi-thin free-floating sections
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   Generation of cryosections
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   Generation of paraffin-embedded sections
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   Histological staining methods
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   Immunohistochemical staining of tissue sections
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   Indirect immunofluorescence staining of tissue sections
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   Filipin staining of unesterified cholesterol
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  Results
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  Bioinformatic analysis of human and murine arylsulfatase G
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   General bioinformatical analysis of arylsulfatase G properties
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   Identification of residues critical for arylsulfatase G activity
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   Disulfide bridges in arylsulfatase G
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   Conservation of arylsulfatase G residues
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   Tissue-specific expression, coexpression and interaction analysis of ARSG
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   Population genetics
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  The human arylsulfatase G
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   ARSG antibody analysis
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   Structural analysis of human arylsulfatase G
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   Analysis of human arylsulfatase G mutants
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  The murine arylsulfatase G
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   Expression of murine arylsulfatase G
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   Transport and targeting of murine arylsulfatase G
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   Maturation of murine arylsulfatase G
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   Glycosylation of murine arylsulfatase G
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  Desulfation of sulfated thyroid hormones by arylsulfatases
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   Analysis of retention volumes of thyroid hormone sulfate standards
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   Desulfation of thyroid hormones by lysosomal sulfatases
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  The arylsulfatase G knockout mouse
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   Arylsulfatase G knockout validation
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   Phenotype of Arsg knockout mice
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   Identification of the storage material
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   Secondary pathologies in Arsg knockout mice
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  Arylsulfatase G in the degradation of heparan sulfate
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   ARSG desulfates the 3-O-sulfate group of N-sulfoglucosamine 3-O-sulfate
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   Inhibition of arylsulfatase G by monosaccharides
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   Substrate specificity of arylsulfatase G
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   Arylsulfatase G activity assay
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   Desulfation of glucosamine 3-O-sulfate derivatives by arylsulfatase G
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   Determination of the pH-optimum of arylsulfatase G
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   Desulfation of N-acetylglucosamine 3-O-sulfate
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   Arylsulfatase G and N-unsubstituted glucosamines
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   Degradation of heparan sulfate with 3-O-sulfate groups
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  Discussion
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  Targeting, maturation and molecular structure of arylsulfatase G
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   Bioinformatical and expression analysis of arylsulfatase G
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   Maturation of arylsulfatase G
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   Targeting of arylsulfatase G
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   Analysis of human arylsulfatase G mutations
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   Conclusions
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  The arylsulfatase G knockout mouse
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   Thyroid hormones and arylsulfatase G
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   ARSG-deficiency causes a mucopolysaccharidosis
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   Disease onset and macroscopic features in Arsg KO mice
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   Comparative analysis of the primary pathology in Arsg KO mice
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   Behavioural changes in ARSG-deficient mice
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   Analysis of the secondary pathologies in the murine and canine ARSG model
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   Mucopolysaccharidosis IIIE patients
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   Conclusion
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  Arylsulfatase G in the degradation of heparan sulfate
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   The arylsulfatase G activity assay
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   Degradation of glucosamine in heparan sulfate
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   Conclusion
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   Outlook
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   Bibliography
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  Appendix
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  Additional results
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   Bioinformatical analysis of human and murine arylsulfatase G
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   Details of gel filtration analyses
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   Production of human arylsulfatase G in CHO DG44 cells
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   Synthesis of N-acetylglucosamine 3-O-sulfate
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   2-Aminoacridone-labelling of unsubstituted glucosamines
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   Anthranilic acid-labelling of glucosamines monosaccharides
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   Analysis of potential MPS IIIE patients
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   Genetics of ARSG
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   Degree of arylsulfatase G conservation
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   SNPs in the human ARSG gene
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   Additional information
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   Sequences
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   List of Figures
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   List of Tables
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   Abbreviations