The heparin binding properties of the bone morphogenetic protein antagonist gremlin. / N Tatsinkam, Arnold Junior.

2015. 228 p.

Research output: ThesisDoctoral Thesis

Unpublished

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Abstract

Gremlin, a 184 amino acid glycosylated protein, is one of several high-affinity endogenous antagonists of the bone morphogenetic protein (BMP) cytokines including BMPs -2, -4 and -7. Both gremlin and its BMP ligands have been shown to bind tightly to heparan sulfate (HS) polysaccharides present on cell surfaces. However, the role played by HS in the interaction between gremlin and its BMP ligands remains unknown. In this study, the heparin binding site on gremlin was characterised. Since the three-dimensional structure of gremlin remains experimentally unresolved, prediction of its heparin binding site was based on docking calculations using its homology models. Within the primary sequence, the predicted heparin binding site comprises interspersed basic residue clusters of arginines and lysines. Using site-directed mutagenesis, strategic combinatorial substitutions of these basic clusters were made. Six C-Myc-tagged gremlin mutants, MGRs 1 - 6, were cloned and expressed in parallel with a C-Myctagged wildtype gremlin protein. All six gremlin-Myc mutants showed markedly reduced heparin binding compared to the wildtype, using heparin affinity chromatography and a heparin-binding ELISA. This verified the predicted mapping of the heparin/HS binding site on gremlin. Ion-exchange chromatography of these gremlinMyc proteins on sulfopropyl (SP)-Sepharose columns showed comparatively weak binding, thus indicating that the interaction between gremlin and heparin/HS is specific. Using a novel Myc-capture GREM1/BMP-4 double sandwich ELISA, the BMP-4 binding capability of both wildtype gremlin-Myc and mutant MGR5 were demonstrated. Moreover, the addition of either soluble unfractionated heparin or the low molecular weight heparin, tinzaparin, in this ELISA neither markedly promoted nor inhibited the interaction between wildtype gremlin-Myc and BMP-4. Overall, these showed that heparin binding is not essential for BMP-gremlin binding. In order to study the functional activities of three selected gremlin-Myc mutants alongside wildtype, their capabilities to inhibit BMP-SMAD1/5/8 signalling were investigated in two functional assays established in C2C12 myoblastic cell lines. Firstly, SMAD1/5/8 phosphorylation was responsive to both BMPs -4 and -7, but this assay failed to generate a graded phospho-SMAD1/5/8 dose response on a sufficiently consistent basis to allow comparison of these gremlin-Myc proteins. This was resolved in the second assay, which employed a SMAD1/5/8-specific luciferase reporter construct. In this reporter assay, the functional activities of concentrated wildtype gremlin-Myc, MGR5 and MGR6 were confirmed.
Original languageEnglish
QualificationPh.D.
Awarding Institution
Supervisors/Advisors
Award date1 Feb 2015
Publication statusUnpublished - 17 Feb 2015
This open access research output is licenced under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

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