Glypican-1: Structural and functional analysis of the N-glycosylated human protein
Sammanfattning: Glypicans are multifunctional cell surface heparan sulphate proteoglycans co-regulating numerous signalling pathways, and are thereby involved in the control of cellular division, differentiation, and morphogenesis. The heparan sulphate (HS) chains are responsible for many of those biological functions; nevertheless recent studies suggest functional roles for the glypican core proteins in mediating the signalling of various growth factors. Glypican-1 (GPC1) is the predominant HS proteoglycan in the developing and adult human brain. In addition, GPC1 is involved in Alzheimer’s disease and scrapie, among others. There is a shortage of detailed structural knowledge regarding the GPC1 core protein and accordingly, we proposed in this thesis to structurally and functionally characterize the human GPC1 core protein and to elucidate its overall topology with respect to the membrane. First, we determined the crystal structure of the human N-glycosylated GPC1 core protein by the two-wavelength MAD method on a SeMet-substituted protein crystal. The GPC1 structure revealed a quite rigid, cylindrical single-domain all ?-helical fold with three substantial loops. Shortly afterwards, we achieved improvements of GPC1 crystal diffraction properties by controlled crystal dehydration using a humidity control device (HC1b) and generated better electron density for crystals of GPC1, allowing the building of previously disordered parts of the structure. Using small angle X-ray scattering and other biophysical approaches, we found that the GPC1 core protein lies on the membrane in a transverse orientation, directing a surface evolutionarily conserved in GPC1 orthologues towards the membrane, where it can interact with enzymes involved in HS substitution in the Golgi apparatus. Furthermore, the N-linked glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. The EXTL3 protein, a member of the exostosin family, functions mainly as an initiator for HS assembly on the glypicans. We have investigated the spectroscopic and structural characteristics of the catalytic region of EXTL3, which exhibits a quite stable extended monomeric structure with two functional domains containing a majority of ? sheets. Additionally, it was found that catalytic EXTL3 is occupied with N-glycans at least at two sites and these N-glycans seem critical for proper EXTL3 biosynthesis. To precisely determine how the GPC1 core protein regulates HS assembly through interactions with EXTL3, investigations of the GPC1-EXTL3 complexes are ongoing, and some preliminary results are presented here.
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