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Structure of human POFUT2 : insights into thrombospondin type 1 repeat fold and O-fucosylation

Chun-I, Chen. Structure of human POFUT2 : insights into thrombospondin type 1 repeat fold and O-fucosylation. 2012, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10025

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Abstract

Protein O-fucosylation is a post-translational modification found on epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSR). The EGF modification was shown to regulate embryonic development by controlling the ligand specificity of Notch (Chapter 5.1) but the role of protein O-fucosyltransferase 2 (POFUT2) -dependent fucosylation of TSRs is less understood. Nevertheless, recent work on Pofut2 knockout mice found that O-fucosylation of TSRs is essential for restricting epithelial to mesenchymal transition, correct patterning of mesoderm and localization of endoderm, suggesting a critical role of POFUT2 in the early development of the mouse embryo (Du et al, 2010). More than 50 human proteins, located either on the membrane or being secreted, contain the TSR consensus sequence for POFUT2-dependent fucosylation. Together, these proteins regulate a wide variety of biological processes such as anti-angiogenesis, growth factor release or innate immunity response (Chapter 5.4).
In order to better understand O-fucosylation on TSRs we carried out a structural and functional analysis of human POFUT2. Crystal structures of native POFUT2 reveals that the protein folds into GT-B superfamily of glycosyltransferase (Chapter 5.3 and Chapter 6.2). HPLC mass-spectrometry enzyme activity measurements of wild-type POFUT2 show that the full activity of POFUT2 is divalent metal ion dependent (Chapter 6.2) and that KM value of wild-type POFUT2 of GDP-fucose and TSR4 are 10 micromolar and 30 micromolar, respectively. Based on the structure of POFUT2 and activity assays of POFUT2 mutants, we proposed that E54 is the catalytic base. The structure of GDP-fucose in complex with E54A-POFUT2 shows that GDP-fucose locates in a conserved minor cleft on the C-terminal domain. Mutations of conserved residues involved in GDP-fucose interaction abolish POFUT2 activity (Chapter 6.2). The in vivo fucosylation assays of TSR4 mutants (Chapter 6.3) and in vitro fucosylation assays of mini-TSR provide a model of how POFUT2 recognizes a properly folded TSR and transfers fucose on it (Chapter 6.1 and 6.3).
Our experimental findings give insight into the catalytic mechanism and the special structural requirements of POFUT2 modifying a completely folded protein sugar acceptor that transiently forms a protein-protein interaction.
Advisors:Hofsteenge, Jan
Committee Members:Hennet, Thierry and Gut, Heinz
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
Item Type:Thesis
Thesis no:10025
Bibsysno:Link to catalogue
Number of Pages:131 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:49
Deposited On:03 Sep 2012 13:55

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