Characterisation of Prodomains in the GDNF Family

Abstract

Growth Differentiation Factor 15 (GDF15) and Neurturin (NRTN) have wide ranging roles in biology, with potential applications in diseases such as diabetes and obesity. As members of the glial cell line derived neurotrophic factor (GDNF) family, both GDF15 and NRTN signal through the RET receptor. RET signalling is essential in normal development and is facilitated by a distinct co-receptor for each GDNF family member. NRTN was originally identified as a neurotrophic factor and is associated with the normal development and maintenance of the nervous system. Additionally, NRTN has positive effects on hyperglycaemia in diabetic animal models. GDF15 is a stress regulator of energy homeostasis and body weight and is associated with numerous pathological conditions including cancer, obesity, and anorexia.

The GDNF family is part of the larger TGF-β superfamily of growth factors. Like all TGF-β superfamily members, proteins are produced as larger precursors consisting of an N-terminal prodomain and a C-terminal mature domain – pro-TGF-β proteins. The mature domains dimerise via disulfide bonds to form active ligands. Furthermore, the prodomain can be cleaved from the mature domain through proteolysis. GDNF family members are cleaved by furin, with a furin site between the pro and mature domains. Prodomains are known to alter TGF-β protein properties including reducing bioactivity and enhancing half-life.

To determine the impact of the prodomain on signalling, the different protein forms were evaluated using cell-based bioactivity assays. Both uncleaved pro-NRTN and pro-GDF15 have reduced RET signalling activity in comparison to their mature domains alone. However, cleaved pro-NRTN becomes as active as mature NRTN. Pro-GDF15 cleavage resulted in a hemi-cleaved protein – with only one prodomain of the dimer cleaved from its mature domain. This hemi-cleaved form of GDF15 was also less active than mature GDF15. Finally, H202D is a common variant of GDF15 in humans. Here, we demonstrate the H202D variant has no impact on GDF15 bioactivity but can affect the detection of the protein by anti-GDF15 antibodies.

Structural characterisation through circular dichroism (CD) reveals both the NRTN and GDF15 prodomains to be largely unstructured, unlike other TGF-β proteins. The prodomain of GDF15 also interacts with heparan sulfate (HS) as demonstrated in gel shift experiments. This may explain the localisation of pro-GDF15 at the cell surface where HS is common. Utilising hydrogen deuterium exchange mass spectrometry (HDX-MS) no interaction between the NRTN prodomain and mature domain could be identified, again unlike other TGF-β proteins. This work highlights the diversity of prodomain structure and function in the TGF-β superfamily and suggests a common theme of unstructured and inhibitory prodomains for the GDNF subfamily.