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Introduction
Analysis of cathepsin H-deficient mice
and identification of a dense cluster
of placenta specific peptidase genes









Inaugural-Dissertation
Zur
Erlangung der Doktorwürde
Der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau

















Vorgelegt von
Tanoh Martin Kouadio
aus Grand-Bassam
(Côte d’Ivoire)
2004
IIntroduction
Dekan der Fakultät für Biologie : Prof. Dr. G. Fuchs
Leiter der Arbeit und Referent: Prof. Dr. A. E. Sippel
Betreuer der Arbeit: Prof. Dr. C. Peters


Tag der Verkündung des Prüfungsergebnisses: 03. November 2004
IIntroduction
N’écouter que son coeur
Et vivre jusqu´au bout
Sa légende personnelle
Quelqu´en soit le prix,
Quelqu´en soit le temps
Que cela pourrait coûter.

Paulo Coelho










































II Introduction
I would like to give special thanks Prof. Dr. Christoph Peters for accepting me in her
laboratory and allowing me to do this work.

My gratitude goes to Prof. Dr. A. E. Sippel for supervising of my work and for helping during
the difficult moments.


Specials thanks to Dr. Jan Deussing and Dr. Thomas Reineckel with whose exceptional daily
supervision I was able to complete this work


Many thanks also go to all the personnel of the Peter´s Lab for their kind collaboration during
my stay.

Mes sincères remerciements Miss Brigitte Kost pour sa très éfficace intervention.
























IIIIntroduction














A ma mère chérie,
Ta patience n´aura pas été
Et ne sera jamais vaine









A Nina et Ama,
Désormais j´aurais
Plus de temps.

IVIntroduction
TABLE OF CONTENTS
1 INTRODUCTION............................................................................................................ 1
1.1 Proteolysis.................................................................................................................. 1
1.2 The Lysosomal and Endosomal Compartment .......................................................... 1
1.3 Lysosomal Proteases..................................................................................................2
1.4 Lysosomal Cysteine Proteases...................................................................................3
1.5 Physiological Roles of Lysosomal Cysteine Proteases.............................................. 4
1.6 Pathological Roles......................................................................................................4
1.7 Placenta-specific Cysteine Proteases: "Cathepsin-J-like Enzymes" .......................... 5
1.8 Gene Targeting...........................................................................................................5
1.8.1 Homologous Recombination and Generation of knock-out Mice ..................... 6
2 research statement 9
3 Materials and methods .................................................................................................. 10
3.1 Materials................................................................................................................... 10
3.1.1 Materials for Molecular and Cell Biology ....................................................... 10
3.1.2 Blotting Material..............................................................................................10
3.1.3 Enzymes, Standards and Antibiotics................................................................
3.1.4 Kits for Nucleic Acid ....................................................................................... 11
3.1.5 Radioactive Substance......................................................................................11
3.1.6 Antibody...........................................................................................................11
3.1.7 Substrat.............................................................................................................
3.1.8 Medium............................................................................................................11
3.1.9 Stamm Solution................................................................................................12
3.1.10 Medium for Bacteria........................................................................................
3.1.11 Vector...............................................................................................................12
3.1.12 Bacteria
3.1.13 ES-Cells12
3.1.14 Mouse Strains...................................................................................................13
3.1.15 Material for mouse methods.............................................................................
3.1.16 Material for Histology...................................................................................... 13
3.1.17 Material for LacZ Staining............................................................................... 13
3.1.18 Pac Isolation and Contig Construction............................................................. 13
3.2 Methods.................................................................................................................... 14
3.2.1 Methods of molecular biology ......................................................................... 14
3.2.2 Hybridization....................................................................................................22
3.3 Biochemical Methods...............................................................................................24
3.3.1 Preparation of Lysosomal Extracts ..................................................................
3.3.2 Determination of Protein Concentration “Bio-Rad DC Protein Assay” .......... 25
3.3.3 Cathepsin H Activity........................................................................................ 26
3.3.4 SDS-polyacrylamide Gel Glectrophoresis (SDS-PAGE) of Proteins.............. 26
3.3.5 Transfer of Protein on to Nitrocellulose and Immunodetection (Western Blot)
27
3.4 Embryonic stem (ES) cell culture ............................................................................ 27
3.4.1 Selection of ES-cell clones............................................................................... 27
3.4.2 Trypsinization and Freezing of HM-1 ES Cells............................................... 28
3.5 Mouse Methods........................................................................................................28
3.5.1 Superovulation induced by Hormonal Manipulation .......................................
3.5.2 Mouse Mating..................................................................................................29
3.5.3 Plug Check.......................................................................................................
3.6 Histology.................................................................................................................. 29
VIntroduction
3.6.1 Dehydratation of Histological Samples............................................................ 29
3.6.2 Embedding of Tissues in Paraffin .................................................................... 30
3.6.3 Tissue Sections.................................................................................................30
3.6.4 Tissue Staining
3.7 LacZ-staining...........................................................................................................31
3.8 Screening of Mice ....................................................................................................
3.9 Statistical Analysis...................................................................................................33
4 Results ............................................................................................................................. 34
4.1 Gene Targeting and Generation of Cathepsin H-Deficient Mice............................. 34
4.2 Functional Inactivation of the Cathepsin H gene ..................................................... 35
4.2.1 Northern-blot Analysis.....................................................................................35
4.2.2 Western-Blot 36
4.2.3 Cathepsin H Enzyme Activity Assay............................................................... 37
4.3 Phenotype of Cathepsin H-Deficient Mice. ............................................................. 38
4.4 Localization of mCathepsin H Expression 39
4.4.1 mCTSH Expression in Lung ............................................................................ 40
4.4.2 Expression in heart and brain ........................................................................... 41
4.4.3 mCTSH Expression in kidney.......................................................................... 41
4.5 Expression of Cathepsin B (CTSB) and L (CTSL) in cathepsin H-deficient mouse.
41
4.6 Screening of cathepsin H-deficient mice ................................................................. 44
4.6.1 Biochemical Metabolite Screen.......................................................................44
4.6.2 Allergy Screen..................................................................................................
4.6.3 Lysosomal Enzyme Screen..............................................................................44
4.6.4 Clinical-Chemical Screen.................................................................................
4.6.5 Immunology Screen.........................................................................................46
4.7 Histological Analyses...............................................................................................47
4.8 Characterization of a Dense Cluster of Placenta-Specific Cysteine Peptidase Genes
and related Genes on Mouse Chromosome 13..................................................................... 49
5 Discussion........................................................................................................................ 53
5.1 Cathepsin H..............................................................................................................53
5.2 Synthesis and Processing of Cathepsin H ................................................................ 53
5.3 Crystal Structure of Cathepsin H ............................................................................. 54
5.4 Catalytic Activity of Cathepsin H ............................................................................ 54
5.5 Tissue Distribution and Functions of cathepsin H ................................................... 55
5.6 Cathepsin H Deficient Mice..................................................................................... 56
6 Summary.........................................................................................................................58
7 References ....................................................................................................................... 59
8 Abbreviation ................................................................................................................... 64
9 Appendix ......................................................................................................................... 65
9.1 Data of metabolite screens ....................................................................................... 65
9.1.1 statistical analysis of metabolite screens.......................................................... 68
9.1.2 Few Box-plot of metabolite screen ................................................................. 69
9.1.3 Data of Allergy screen...................................................................................... 70
9.1.4 Box-plot of Allergy screen............................................................................... 71
9.2 Lysosomal screen.....................................................................................................72
9.2.1 Data of lysosomal screen..................................................................................
9.2.2 Statistical box-plot of lysosomal screen........................................................... 73
9.3 Clinical chemical screen........................................................................................... 74
9.3.1 Data of clinical chemical screen....................................................................... 74
VIIntroduction
9.3.2 Statistical analysis of the data of clinical chemical screen............................... 75
9.3.3 Few statistical box-plot of clinical-chemical screen ........................................ 76
9.4 Data of immunological screen.................................................................................. 77
9.4.1 Elisa measure: Antibody TBF β........................................................................ 77
9.4.2 easure: Antibody aK............................................................................. 77
9.4.3 Elisa measure: Antibody Ig3............................................................................ 78
9.4.4 easure: Antibody IgA ........................................................................... 78
9.4.5 Elisa measure: Antibody TGF β 79
9.4.6 easure: Antibody Rheuma .................................................................... 79
9.4.7 Statistical analysis of imunological elisa data.................................................. 80
9.4.8 Statistical box-plot of ................................................. 81
9.5 Data of immunology screen ..................................................................................... 82
9.5.1 Statistical analysis of imunological Facs data 87
9.5.2 Few box-Plots of umunological Facs............................................................... 88


VIIIntroduction
1 INTRODUCTION
1.1 Proteolysis
Proteolysis is the hydrolytic cleavage of peptide bonds with formation of smaller
polypeptides. This extremely regulated, ubiquitous, and controlled process is performed by
proteolytic enzymes. Proteolysis can be accomplished either in a complete and nonspecific
manner called "bulk" proteolysis or by a limited mechanism in which specific peptide bonds
of protein are selectively splitted (Wolf 1992). The type of proteolysis mechanism performed
in the eukaryote cells depends on the substrate, on its localization, and on the contents of
proteolytic enzymes in the specific compartment (Barrett 1992).

1.2 The Lysosomal and Endosomal Compartment
The lysosome is the compartment of the cell in which the terminal process of degradation of
proteins is accomplished (deDuve 1963). Polysaccharides, lipids, nucleic acids and proteins
are submitted to the action of approximately 40 hydrolytic enzymes, like proteases, nucleases
as well as lipases that are located within the lysosomes. The lysosomal enzymes exhibit a pH
optimum between pH4.5 and pH5.5 in agreement with the acidic medium of the lysosome.
The low pH value in the lysosomes, compared to the cytosol (pH 7,2), is maintained by the
protons that are driven in the lysosomes by the proton pumps located in the lysosomal
membrane (Cuppoletti, Aures-Fischer et al. 1987). Lysosomal membranes also contain carrier
proteins allowing transport of final degradation product into the cytosol like sugar,
nucleotides, fatty acids and amino acids (Kornfeld and Mellman 1989). Lysosomal enzymes
are synthesized as praeproenzymes on the rough endoplasmatic reticulum (ER). Those
präproenzymes consist of characteristic domains: a aminoterminal signal peptide of 16-18
amino acids, a propeptid of 62-100 amino acids and the catalytic active, mature enzyme
region of 220-230 amino acids (Kirschke 1995). The newly synthesized polypeptide is
transferred with the help of the hydrophobic amino terminal signal sequence into the rough
endoplasmatic reticulum (ER) lumen. The propeptide prevents a premature proteolytic
activity and ensures the stability of the proenzyme (Coulombe, Grochulski et al. 1996 ;
Cygler, Sivaraman et al. 1996; Groves, Taylor et al. 1996). In the lumen of the ER occurs the
1Introduction
cleavage of the signal peptide and glycolysation of the new protein by fixation of an N-linked
oligosaccharide at a specific Asn residue in the recognition sequence Asn-X-Ser/Thr.
After transfer to the cis-Golgi region one or more mannose residues of the oligosaccharide
become phosphorylated (Pohlmann, Waheed et al. 1982). The phosphorylation of mannose
residues occurs in two step catalyzed by two different enzymes. In the first step, the transfer
of GlcNAc-phosphate from UDP-GlcNAc is catalyzed by a UDP N-Acetylglucosamine, the
lysosomal enzyme 1-phosphotransferase (GlcNAc-phosphotransferase). In the second step,
catalyzed by a phosphodiester, GlcNAcase or uncovering enzyme (UCE), the covering
GlcNAc is removed exposing mannose 6-phosphate (von Figura 1986). This mannose 6-
phosphate (M6P) moving process is followed by the binding of the propeptide to the luminal
domain of M6P receptors in the trans-Golgi region. The membrane, containing M6P receptor
and the proenzyme bound into vesicles, fuse with a sorting vesicle called late endosome. The
acidic medium of the endosome allows the propeptide to separate from the M6P receptor. In
addition, a phosphatase within the late endosomes prevents the lysosomal enzymes from
binding to the M6P receptor which recycles to the trans-Golgi region or occasionally to the
cell surface while the proenzyme is transported to the lysosomes (von Figura 1986; von
Figura 1986; von Figura 1987).
Within the lysosome the process of maturation, which consists of removing the propeptide by
limited proteolysis, is often followed by a cleavage of the mature enzyme into a light and a
heavy chain connected by disulphide bonds (for example: Cathepsin B, H and L).
All these processing steps which lead to mature and active lysosomale enzymes can be
already accomplished in a prelysosomal compartment like the endosomes (Hasilik 1992).
1.3 Lysosomal Proteases
The lysosomal/endosomale compartment contains a rich selection of proteolytic enzymes
among them an important number of proteases for the complete and also limited degradation
of proteins. Proteases comprise endopeptidases, which hydrolyze peptide bonds within a
polypeptide chain, and exopeptidases, which cleave proteins only near the C-or N- terminus.
Certain proteases display both endopeptidase and exopeptidase activities and are classified by
convention as endopeptidase. Protein breakdown mediated by lysosomal proteinases is
presumed to be initiated by endopeptidases, which are rate limiting. The process is continued
by lysosomal exopeptidase which are extremely active in the degradation of even quite large
peptide (Barrett 1992). Many dipeptides, can diffuse through the membrane for final
hydrolysis by dipeptidases elsewhere in the cell (Kirschke 1987).
2

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