Glypican-1: Structural and functional analysis of the N-glycosylated human protein

Abstract

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 α-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 β 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.

Abstract (Swedish)

Popular Abstract in English

All living things somehow communicate with each other. Cell-to-cell communication, or signalling, occurs on the molecular level, regulating the body’s activities and coordinating various cell actions and is thereby valuable for realizing cell as well as system functions. Errors in the cellular transferred information may induce diseases such as cancer and autoimmune diseases, among others. In fact, most diseases enclose at least one malfunction in cell communication pathways. Understanding the cellular signalling pathways and the components involved would be significant for effective treatment of those diseases. Many components of the extracellular environment, in particular the cell surface receptors, enable the cells to recall the extracellular signals and then trigger intracellular chains of biochemical events creating the response. These receptors often use heparan sulphate proteoglycans (HSPG) to promote and control ligand binding and activation, due to the interactions of HSPG core proteins and/or the heparan sulphate (HS) chains with the ligands.

Glypican (GPC) is a family of HSPG proteins that are anchored to the external leaflet of the cell membrane where they interact with several extracellular ligands and receptors and therefore act as mandatory co-receptors. GPCs are involved in the regulation of many biological processes such as cellular adhesion, division, differentiation and morphogenesis. The HS chains are responsible for many of these biological functions, but recent studies suggest functional roles for the GPC core proteins in mediating various morphogen and growth factor signalling.

Glypican-1 (GPC1) is one of the six members of the vertebrate GPC family that is mainly expressed in the neural and skeletal systems during development and ubiquitously in the adult. GPC1 is involved in the uptake of different macromolecules such as growth factors, viral proteins, polyamines and cytokines. Many reports concluded that GPC1 is important for brain development and function, and further revealed its involvement in the pathogenesis of several neurodegenerative diseases and glioma, pancreatic and breast cancers. Unfortunately, there is a shortage in structural knowledge about the GPC core proteins. The overall objective of this thesis is to structurally characterize the GPC1 core protein and its overall topology with respect to the cell surface. This will be of great assistance to gain insights into the functional roles of GPC1 and the mechanism behind HS assembly on their core proteins.

I hope that I have written this dissertation at a level at which readers with scientifically diverse backgrounds can understand and appreciate it. First there is a general introduction about the proteins and their post-transcriptional modifications focusing on the HS chains biosynthesis via the exostosin family enzymes. In the second chapter there is a brief, but sufficiently detailed description of the HSPG, in particular the GPC protein family and their roles in modulating various signalling processes. Afterwards, I will try to summarize, in chapter three, the available functional and biochemical knowledge regarding the GPC1 proteoglycan. Chapter four introduces an investigation of the current study followed by concise description of the methods that were used. Finally, chapter five pinpoints the main findings of the papers included in this dissertation and discusses further future directions.

• author: Abdelhady, Wael Awad
• supervisor: Derek Logan; Katrin Mani
• opponent: Prof Savvides, Savvas, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium.
• organization: Biochemistry and Structural Biology
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Biological Sciences
• keywords: Proteoglycans, glypicans, glypican-1, heparan sulphate, N-glycosylation, exostosin-proteins, X-ray crystallography, crystal dehydration, diffraction anisotropy, Small angle X-ray scattering (SAXS9, protein spectroscopy
• pages: 149 pages
• defense location: Kemicentrum, Lecture Hall C
• defense date: 2015-05-28 09:30:00
• ISBN: 978-91-7422-399-6
• language: English
• LU publication?: yes
• id: 7d0d8f43-d852-41b5-92bc-16de7b4d446a (old id 5336702)
• date added to LUP: 2016-04-04 11:08:29
• date last changed: 2018-11-21 21:02:55