Dissertation/Thesis Abstract

Die extrazelluläre Domäne der humanen Guanylatzyklase C und ihre Liganden: Rekombinante Expression, strukturelle Charakterisierung und Aktivitätsbestimmung
by Weiglmeier, Philipp Richard, Ph.D., Universitaet Bayreuth (Germany), 2014, 228; 27610511
Abstract (Summary)

The interaction of intestinal guanylyl cyclase C (GC-C) with the peptide hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin STh is of eminent importance for the maintenance of electrolyte and water homeostasis in the small intestine. Guanylin, uroguanylin and STh are peptides with a length of 15 to 19 amino acids and are rich in cystines. The binding of these peptides to the extracellular domain (ECD) of GC-C activates its intracellular guanylyl cyclase domain and triggers a cGMP-mediated secretion of anions into the intestinal lumen. GC-C is a promising target for innovative therapeutic strategies. This is illustrated by the role of the STh/GC-C interaction in acute secretary diarrheal diseases caused by enterotoxigenic Escherichia coli (ETEC) infection as well as by the recent finding that GC-C activation by its ligands is correlated with a diminished incidence of colorectal carcinoma. In order to contribute to a better understanding of the molecular basis of this receptor/ligand interaction and to lay the foundations for further binding studies, efficient protocols for the recombinant expression and purification of the GC-C agonists uroguanylin and STh were developed in the present work. Furthermore, several recombinant expression systems, based on Escherichia coli, the yeast Pichia pastoris as well as mammalian cell lines, were explored with respect to their suitability to express the entire ECD of GC‑C or a receptor fragment that is able to bind GC-C's ligands. In vivo, guanylin and uroguanylin are expressed as pro-hormones. The role of the pro-peptide is to stabilize the biologically active conformation of the hormones and thereby to render possible the formation of the native disulfide linkages. The hormones are released from the respective pro-hormones by proteolytic cleavage. As a first step, uroguanylin was produced by recombinant expression, purification and tryptic digestion of the precursor protein prouroguanylin. NMR spectroscopy could show that the purified, recombinant peptide had chemical shifts consistent with biologically active uroguanylin. The presence of uroguanylin's two disulfide bonds was confirmed by mass spectrometry. These findings showed that the purified uroguanylin adopts its native conformation. For the recombinant expression of biologically active heat-stable enterotoxin STh its considerable structural similarity to uroguanylin was used and a chimeric precursor protein consisting of the N‑terminal pro-peptide of prouroguanylin and the amino acid sequence of STh was developed. This novel fusion protein was termed Prouro-STh. The formation of the native disulfide bond pattern of STh should be supported in the context of the fusion protein Prouro-STh in a way very similar to the situation in prouroguanylin. Using this approach, recombinant STh could be obtained in good yield and purity and was subsequently characterized (Weiglmeier et al., 2013). The biological activity of both recombinant peptides, uroguanylin and STh, was confirmed by an in vitro activity assay. The expression system established in the present work can be used for the production of other GC-C agonists derived from uroguanylin and STh. This has been shown by the successful expression and purification of the mutant variants uroguanylin D3R D6E, STh L9Y and STh Y5R L9Y. The pro‑peptide tolerates the newly introduced point mutations in its target, i. e. the uroguanylin or STh, without any apparent impediment on pro-peptide mediated folding. In the second part of this work the receptor fragment MiniGC-C was expressed in E. coli. MiniGC‑C has a length of 197 amino acids and corresponds to the membrane proximal subdomain of the ECD. MiniGC-C is a folded soluble protein that can be purified at a high yield. However, NMR titration experiments were unable to confirm any interaction between MiniGC-C and uroguanylin or STh, respectively. This observation leads to the conclusion that, despite predictions to the contrary, the interaction between the ligands and the ECD is not solely mediated by the latter's membran proximal subdomain. Several protocols for the expression of the entire ECD of GC-C in either E. coli, the methylotrophic yeast Pichia pastoris or mammalian cell culture were developed and evaluated. The constructs Trx-ECD-GC-C and NusA‑Cys‑‑ECD‑GC‑C, which were used for expression in E. coli, were not suitable for ligand binding. While Trx-ECD-GC-C was completely unsoluble the NusA‑Cys‑‑ECD‑GC‑C fusion protein turned out to be misfolded and formed non-native oligomers. The ECD was expressed in P. pastoris and mammalian cell culture. However, the yield of expressed protein in both systems was low. Moreover, both constructs were quite unstable and were lost due to degradation during purification. Although it was not possible to obtain a soluble well-folded and pure fragment of GC-C which retains the ability to bind GC-C's ligands the purification protocols evaluated in the present work offer nevertheless a basis and novel starting point for future efforts to achieve this goal. It should be highlighted that the relatively easy and inexpensive method for production and isotopic labeling of agonists of GC-C developed here is an important tool for the NMR spectroscopic characterization of receptor/ligand interactions involving GC-C. Because of the recent focus on the pharmacological potential of uroguanylin and STh variants the results presented in this work constitute an important contribution to the development of new active pharmaceutical ingredients.

Indexing (document details)
Advisor:
Commitee:
School: Universitaet Bayreuth (Germany)
School Location: Germany
Source: DAI-C 81/4(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry, Chemistry, Physiology
Keywords: Intestinal guanylyl cyclase C, Peptide hormones, Electrolyte homeostasis, Water homeostasis, Small intestine
Publication Number: 27610511
ISBN: 9781392620656
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