The human saposins are four homologous activator proteins that are essential for the lysosomal degradation of sphingolipids (SLs) with small headgroups. They function in part to increase the solvent accessibility of these SLs to specific acid hydrolases by two proposed modes of action. In the solubilizer model, saposins extract SLs into soluble saposin-lipid complexes, and in the liftase model, the saposins reorganize lipids within membranes. The four saposins, called saposins A, B, C and D, have dual characters and exist as soluble proteins and in membrane-associated states. In this thesis, I first present the crystal structure of saposin D in a lipid-free state. The structure exhibits a closed, monomeric fold as previously described for saposins A and C. Next, I examine the lipid interaction properties of saposin A and determine a crystal structure of SapA in a lipid-bound complex. The complex forms a discoidal lipoprotein particle composed of highly ordered bilayer-like hydrophobic core surrounded by a protein belt consisting of two copies of saposin A in an open conformation. The saposin A-lipid discs are most likely the effective substrate-presenting particles in galactosylceramide hydrolysis. Finally, I compare the lipid interaction properties of the four saposins and address membrane perturbation, liposome binding, lipid solubilization and lipoprotein particle formation for each protein. Each saposin displays a unique behavior in the presence of liposomes under conditions that mimic the lysosomal environment. In particular, saposin D reveals simultaneous formation of different sized protein-lipid complexes, which appear to be primarily dependent on the lipid to protein molar ratio. A comparison of the available structures of saposins A, B and C in the “closed” and “open” conformations reveals structural hinge regions that likely shape the different types of saposin self-association. These states are directly related to the protein-lipid solubilization and/or membrane association properties of the saposins. Collectively, these findings present a more complete understanding of the lipid interaction properties of the saposin proteins and provide new insights into their role as activator proteins.

The human saposins are four homologous activator proteins that are essential for the lysosomal degradation of sphingolipids (SLs) with small headgroups. They function in part to increase the solvent accessibility of these SLs to specific acid hydrolases by two proposed modes of action. In the solubilizer model, saposins extract SLs into soluble saposin-lipid complexes, and in the liftase model, the saposins reorganize lipids within membranes. The four saposins, called saposins A, B, C and D, have dual characters and exist as soluble proteins and in membrane-associated states. In this thesis, I first present the crystal structure of saposin D in a lipid-free state. The structure exhibits a closed, monomeric fold as previously described for saposins A and C. Next, I examine the lipid interaction properties of saposin A and determine a crystal structure of SapA in a lipid-bound complex. The complex forms a discoidal lipoprotein particle composed of highly ordered bilayer-like hydrophobic core surrounded by a protein belt consisting of two copies of saposin A in an open conformation. The saposin A-lipid discs are most likely the effective substrate-presenting particles in galactosylceramide hydrolysis. Finally, I compare the lipid interaction properties of the four saposins and address membrane perturbation, liposome binding, lipid solubilization and lipoprotein particle formation for each protein. Each saposin displays a unique behavior in the presence of liposomes under conditions that mimic the lysosomal environment. In particular, saposin D reveals simultaneous formation of different sized protein-lipid complexes, which appear to be primarily dependent on the lipid to protein molar ratio. A comparison of the available structures of saposins A, B and C in the “closed” and “open” conformations reveals structural hinge regions that likely shape the different types of saposin self-association. These states are directly related to the protein-lipid solubilization and/or membrane association properties of the saposins. Collectively, these findings present a more complete understanding of the lipid interaction properties of the saposin proteins and provide new insights into their role as activator proteins.

Fabry disease is an inherited defect of lysosomal α-galactosidase A (α-GALA), causing progressive accumulation of glycosphingolipids with terminal α-galactosyl moieties, especially globotriaosylceramide (Gb3Cer) and in to a small extent also galabiosylceramide (Ga2Cer) and blood group B glycolipids, in most tissues and body fluids. This diploma thesis is an extension of previous laboratory studies and intends to contribute to clarification of some specific features of catabolic pathways of glycolipids substrates in lysosomal storage disorders, especially blood group B glycolipids. Therefore, analysis of human pancreas and lungs tissues was performed using TLC imunodetection and immunohistochemical analysis of these glycolipids. The most striking observation was massive accumulation of B-6-2 glycolipid and of others complex B-glycolipids in the pancreas of the patient with Fabry disease with blood group B. The level of blood group B substrates exceeded significantly storage of Gb3Cer substrate. An important part of this work were metabolic experiments in cell cultures in order to answer the question about participation of related glycosidases - α-galactosidase A and α-N- acetylgalactosaminidase (α -NAGA) in the lysosomal degradation of glycosphingolipids with terminal α-galactose. Loading experiments were...

Membrane proteins are important targets that represent more than 50% of current drug targets. However, characterisation of membrane proteins falls behind compared to their soluble counterparts. The most challenging part of membrane protein research is finding a suitable membrane mimetic that stabilises them in solution and maintains their native structure and function. The recently developed saposin-A (SapA) based lipid nanoparticle system seems to be advantageous over existing membrane mimetic system. It provides a native-like lipid bilayer, high incorporation yield and more importantly size adaptability. SapA lipid nanoparticles have been applied to structural studies and two high-resolution structures of membrane proteins were previously obtained using cryo-electron microscopy. This thesis aimed to study small-to-medium sized membrane proteins in SapA lipid nanoparticles using NMR spectroscopy. We first explore the mechanism of SapA lipid nanoparticle formation for the purpose of establishing an incorporation protocol that can be applied to most membrane proteins. The effect of pH and the presence of detergents on the opening of SapA was investigated in Chapter 2. A proposed energy diagram describing the mechanism of SapA opening is reported with which we were able to develop a protocol that can generate different sizes of SapA-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) nanoparticles. In addition, we also showed that SapA can form lipid nanoparticles with various lipid compositions, showing the versatility of the system. In Chapter 3, we validated the ability of SapA lipid nanoparticles to be used as a membrane mimetic. A -barrel model protein, bacterial outer membrane protein X (OmpX), was incorporated into SapA-DMPC nanoparticles and a 2D 15N-1H correlation NMR spectrum was recorded. Our result was compared to the NMR parameters of the same protein in MSP nanodiscs from the literature, and it was concluded that SapA lipid nanoparticles indeed provide a lipid bilayer environment similar to MSP nanodiscs. Because of high incorporation yield, we were able to incorporate OmpX into different lipid compositions to investigate the effect of lipid head groups and aliphatic chains on the membrane protein's chemical environment. Next, the applicability of SapA lipid nanoparticles was expanded to -helical transmembrane proteins in Chapter 4. Two microbial rhodopsins, Anabaena sensory rhodopsin (ASR) and Natronomonas pharaonis sensory rhodopsin II (pSRII), were tested. The parameters for expression and purification of ASR were first screened for the optimal yield. Although incorporation of ASR resulted in inhomogeneous particles due to imperfect experimental procedure, pSRII in SapA-DMPC nanoparticles showed high sample quality. The 2D NMR spectrum of pSRII in SapA-DMPC nanoparticles shows distinct differences to pSRII in detergent micelles, suggesting substantial effects from the membrane mimetic on the conformation of the membrane protein. Despite the good NMR spectral quality considering the large particle size, perdeuteration of pSRII and the lipids will be necessary for further investigation. With the SapA lipid nanoparticles established, we aimed to use it for the study of a biologically important G protein-coupled receptor, 1-adrenergic receptor (1AR), discussed in Chapter 5. The possibility of expressing 1AR using a cell-free expression system was explored first. Although a good amount of the protein was obtained, only a fraction of it was functional. Therefore, a conventional baculovirus-insect cell expression system was used to produce selective isotope labelled 1AR for NMR studies. NMR spectra of 1AR in SapA-DMPC nanoparticles with activating ligands and an intracellular binding partner were recorded and compared to the spectra of the same protein in detergents. This revealed a more active-like conformation of ligand-bound 1AR in the lipid bilayer, suggesting that certain parts of the protein are sensitive to the membrane mimetic used. This emphasises the importance of using a native-like membrane mimetic to capture the full properties of membrane proteins. In conclusion, I demonstrate in this thesis that SapA lipid nanoparticles are a versatile membrane mimetic system that can accommodate membrane proteins with different sizes and folds. This system is also compatible with solution NMR spectroscopy enabling structure and dynamics studies of biologically important membrane proteins. We believe SapA lipid nanoparticles will have a significant impact on membrane protein research in the future.

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