Structural analysis of human growth hormone with respect to the dominant expression of GH mutations in isolated growth hormone deficiency type II [Elektronische Ressource] / vorgelegt von Daniel I Iliev

Daniel I. Iliev

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• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2005
• Nombre de lectures: 8
• Langue: English

Extrait

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Aus der Universitätsklinik für Kinder- und Jugendmedizin Tübingen Abteilung Kinderheilkunde I mit Poliklinik Ärztlicher Direktor: Professor Dr. D. Niethammer Sektion Pädiatrische Endokrinologie Leiter: Professor Dr. M.B. Ranke

 Structural Analysis of Human Growth Hormone with Respect to the Dominant Expression of GH Mutations in Isolated Growth Hormone Deficiency Type II 

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Inaugural-Dissertation zur Erlangung des Doktorgrades der Medizin

der Medizinischen Fakultät der Eberhard-Karls-Universität zu Tübingen

vorgelegt von Daniel I Iliev  aus  Sofia, Bulgarien  2005

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Dekan:

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Professor Dr. C. D. Claussen

1. Berichterstatter: Privatdozent Dr. G. Binder

2. Berichterstatter: Privatdozent Dr. R. Lammers

 

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Für meine Snejina

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Parts of this thesis have already been published in:

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Iliev DI, Wittekindt NE, Ranke MB, BinderG 2005 Structural Analysis of

Human Growth Hormone with Respect to the Dominant Expression of GH

Mutations in Isolated Growth Hormone Deficiency Type II. Endocrinology 146:

1411-1417

 

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Contents  1. Abbreviations  p. 6 2. Abstract  p. 7 3. Introduction  p. 9 4. Aim of the study  p. 16 5. Materials and methods  p.17 5.1. DNA Vectors  p.17 5.2. Cell culture  p. 19 5.3. Transfection experiments  p.19 5.4. RNA extraction and Real-time RT-PCR  p. 20 5.5 hGH and IGFBP-2 measurements p. 20  5.6. Western blot analysis  p. 20 5.7. Cell counting  p. 21 5.8.β-galactosidase and luciferase activities  p. 21 5.9.β-galactosidase staining  p. 21 5.10. Statistical data analysis p. 22  6. Results  p.23 7. Discussion  p. 50 8. Acknowledgements  p. 56 9. Summary  p. 57 10. References  p. 62 11. Curriculum vitae  p. 70

 

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1. Abbreviations:  aa  amino acid AIDA  advanced image data analyser ATCC  American type culture collection cDNA  complementary DNA CE  cell extract Ct threshold cycle  ER  Endoplasmic reticulum ESPE  European Society for Paediatric Endocrinology GH  growth hormone GHD  growth hormone deficiency hGH  human GH IGFBP  insulin-like growth factor binding protein IGHD  isolated growth hormone deficiency M  medium MPHD or CPHD  multiple or combined pituitary hormone deficiency rhGH  recombinant hGH RIA radioimmunoassay  wt  wild type

 

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2. Abstract  Human GH protein consists of four alpha-helices and contains two disulfide bridges. Isolated growth hormone deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations ofGH-1 to the leading disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32-71 which comprise the complete loop between alpha-helices 1 and 2. The mutant GH protein exerts a dominant-negative effect on wild-type (wt) GH secretion by unclear mechanisms. For the study of the structure-function relationship of GH mutants concerning the dominant-negative effect, expression vectors harbouring mutated GH cDNAs (the mutations affecting either the linker region between alpha-helices 1 and 2 or the disulfide bridge Cys182-Cys189) were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH 4 C 1  cells. Plasmids encoding eitherβluciferase or IGFBP-2 were cotransfected with pwtGH-galactosidase, and either of the GH mutants. For the study of a potential dominant-negative effect due to disturbed Zn 2+ - binding, expression vectors harbouring mutated GH cDNAs were constructed in which triplets encoding histidine and glutamine residues were mutated to triplets encoding alanine residues. These plasmids were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH 4 C 1 cells. Compared to the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32-46 or 32-52. Deletion of more aa (32-53, 32-63, 32-69, 32-71) ascendingly decreased GH secretion and content of GH in parallel with the increasing length of the deleted stretch. Disruption of the disulfide bridge constituted between Cys182-Cys189 in the fourth alpha-helix of GH protein (mutant del188-190GH) did not show to play a role in the exertion of the dominant-negative effect. Partial or complete disturbance of Zn 2+ -binding by amino-acid substitutions in the responsible

 

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