Analysis of two human gene clusters involved in innate immunity [Elektronische Ressource] / vorgelegt von Zhihong Wu

Sujets

• Gesundheit

Informations

• Publié par: christian-albrechts-universitat_zu_kiel
• Publié le: 01 janvier 2005
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Analysis of Two Human Gene Clusters
Involved in Innate Immunity







Dissertation

zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel







vorgelegt von

Zhihong Wu



Kiel

Mai 2005

































Referent: Prof. Dr. rer. nat., Dr. h. c. Thomas C. G. Bosch

Korreferent: Prof. Dr. rer. nat. Jens-Michael Schröder

Tag der mündlichen Prüfung: 2005-12-08
Zum Druck genehmigt: Kiel, den 2005-12-21



------------------------------- Der Dekan






Contents



List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . i

1 Introduction 1
1.1 Chemical antibiotics: an unsolved and growing problem .. . . . . . . . . . . . . . . . . .. . . . .1
1.2 The importance of the innate immune response in living organisms . . . . . . . . . . .. . . . .2
1.3 Antimicrobial peptides (AMPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
1.3.1 Discovery of AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3.2 Classification of AMPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
1.3.3 The importance of AMPs in human skin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
1.4 The barrier function of human skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.4.1 The cornified cell envelope (CCE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
1.4.2 The epidermal differentiation complex (EDC) . . . . . . . . . . . . . . . . . . . . . . . . . . .9
1.4.3 The functions of the SFTP family members . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10
1.5 Two emerging novel families of AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . .12
1.6 Objective of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
2 Materials and Methods 16
2.1 Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 17
2.3 Tissues and RNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 18
2.4 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.5 Conventional RT-PCR . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.6 Real-time RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.7 In-silico cloning of the novel human SFTP and SPINK genes . . . . . . . . . . . . . . . . . . 21
2.8 5’ and 3’ Rapid Amplification of cDNA Ends (RACE) . . . . . . . . . . . . . . . . . . . . . . . 23
2.9 Long-distance PCR and the short-range PCR-walking strategy . . . . . . . . . . . . . . . . . 25
iii

Contents iv


2.10 Subcloning and expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 25
2.10.1 Preparation of expression constructs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.10.2 Induction and expression of the polyhistidine-tagged fusion protein . . . . . . 26
2.10.3 Digestion and purification of the recombinant protein . . . . . . . . . . . . . . . . . 28
2.10.4 Protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 28
2.11 Generation of goat antibodies . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.12 Immunoblot analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.13 Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 29
2.14 Enzymatic Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.15 Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3 Results 31
3.1 The human clustered S100 fused-type protein (SFTP) gene family .. . . . . . . . . . . . . 31
3.1.1 Characterization of the full-length human hornerin cDNA . . . . . . . . . . . .. . . . 31
3.1.2 Characterization of three novel SFTP members cDNAs. . . . . . . . . . . . . . . . . . 32
3.1.3 Genomic structure of human SFTP genes. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 34
3.1.4 Tissue distribution of human SFTP expression. . . . . . . . . . . .. . . . . . . . . . . . . 36
3.1.5 Expression of human SFTP genes in skin disease tissues. . . . . . . . .. . . . . . . . . 38
3.1.6 Comparative analysis of human SFTP mRNA-expression in proliferating
and differentiating human keratinocytes . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 41
3.2 Identification of four novel human kazal-type serine protease inhibitor-like
cDNAs (SPINKs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2.1 Characterization of the full-length human LEKTI-2 cDNA. . . . . . . . . . . . . . . 44
3.2.2 Characterization of SPINK9 homologous cDNAs. . . . . . . . . . . . . . . . . . . . . . .. 46
3.2.3 Genomic structure of human SPINK9 and its homologs genes. . . . . . . . . . . . . 50
3.2.4 Expression analysis of the human SPINKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3 Functional analysis of the human hornerin and LEKTI-2 . . . . . . . . . . . . . . . ... . . . . . 57
3.3.1 Purification and Characterization of recombinant hornerin fragments.. . . . . . . 57
3.3.2 Purification and Characterization of recombinant LEKTI-2. . . . . . . . . . . .. . . . 63
3.3.3 Analyses of protease activity of HRNR3. . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . 64

Contents v


3.3.4 Analyses of antimicrobial activity of human hornerin fragments and
LEKTI-2. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . 64
3.3.5 Generation of polyclonal goat antisera against hornerin fragments and
LEKTI-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4 Discussion 70
4.1 The human clustered S100 fused-type protein (SFTP) gene family .. . . . . . . . . . . . . . 71
4.2 The human clustered kazal-type serine protease inhibitor (SPINK) gene family 74
4.3 Structural and functional implications for the novel SFTP/SPINK gene products
based on comparative sequence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.4 The SFTP genes expression upregulated by cross-talking of the mitogenactivated
protein kinases pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.5 The SPINK7-v2: a chimeric protein encoded by two chromosomes . . . . . . . .. . . . . . 86
4.6 The SPINK9 product: LEKTI-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.7 The hornerin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . 90
4.8 The polyclonal goat antisera against hornerin fragments and LEKTI-2 . . . . . . . .. . . . 91
Abstract 92
Zusammenfassung 95
Reference 98
Appendix 110
5.1 Nucleotide and amino acid sequences of human THHL1 cDNA . . . . . . . . . . . … .110
5.2 Nucleotide and aman repetin cDNA. . . . . . . . . . . . . . . . 111
5.3 Nucleotide and aman hornerin cDNA. . . . . . . . . . . . . . . .112
5.4 Nucleotide and aman ifapsoriasin cDNA. . . . . . . . . . . . .113
Erklärung 114
Acknowledgments 115
Curriculum Vitae 116 ListofFigures
1.1 Preparative C8 RP HPLC analysis of heparin bound proteins from pooled
stratumcorneumextractsofhealthyperson’sheelcallus. . . . . . . . . . . . 15
3.1 Schematicstructureofhumanhornerinprotein. . . . . . . . . . . . . . . . . 33