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Linking structure and function of the asialoglycoprotein receptor H1-CRD using site-directed mutagenesis and isotope labeling

Johansson, Anna Karin. Linking structure and function of the asialoglycoprotein receptor H1-CRD using site-directed mutagenesis and isotope labeling. 2007, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8736

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Abstract

The asialoglycoprotein receptor (ASGP-R) is a C-type lectin, abundantly expressed on hepatocytes. It mediates the clearance of desialylated glycoproteins carrying terminal galactose (Gal) or N-acetyl-galactosamine (GalNAc) residues through endocytosis. The receptor consists of two subunits, H1 and H2, both containing a carbohydrate recognition domain (CRD), responsible for ligand binding. Organ specific expression and high ligand specificity makes the ASGP-R a potential candidate for targeted drug delivery to the liver.
The aim of this work has been to improve the understanding of H1-CRD by linking function and structure of the subunit. Site-directed mutagenesis was used to deduce the role of three N-terminal cysteines in dimer formation seen upon expression of H1-CRD in vitro. Step-wise substitution of the cysteines by serines proved to reduce and even completely abolish the dimerization. However, GalNAc affinity of the mutant proteins was impaired as a result of the modification(s).
Site-directed mutagenesis was applied in a second study to investigate the functional importance of selected amino acid residues in the binding site of H1-CRD. Five single mutant proteins were created, identifying one residue of major importance for GalNAc binding. Two other residues displayed only minor influence on ligand binding, while one mutation was seen to result in an improvement of the affinity for GalNAc. In addition, one mutant was created to investigate the role of a histidine vs. a glutamate in pH-dependent ligand binding exhibited by H1-CRD.
Finally, a method for high efficiency isotope labeling of H1-CRD was established. The method was shown to yield protein with high incorporation levels of both C and N,
providing a good basis for a future structure determination of H1-CD by NMR.
Advisors:Ernst, Beat
Committee Members:Spiess, Martin
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molekulare Pharmazie (Ernst)
UniBasel Contributors:Ernst, Beat and Spiess, Martin
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8736
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:169
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:17 Jul 2009 08:57

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