Abstract
Infection with the pathogenic Plasmodium falciparum parasite causes the potentially deadly Malaria disease which leads to over 1 million fatalities each year according to the WHO (World Health Organization). Individuals subjected to multiple infections gradually become immune to the disease symptoms and vaccine research is focused on trying to mimic or advance this immune acquisition. Immunity is primarily caused by acquisition of antibodies directed against a family of Plasmodium protein antigens called PfEMP1s located on the surface of infected erythrocytes. The PfEMP1 proteins are adhesive and allow infected erythrocytes to attach to endothelial cells and tissues. As a consequence infected erythrocytes sequester in the bloodstream, clotting blood vesicles and leading to severe and potentially fatal disease symptoms. The PfEMP1 proteins are multidomain antigens consisting of two types of structural domains termed DBL (Duffy Binding-like) and CIDR (Cysteine-rich Inter-domain Region) arranged in PfEMP1 variants containing from two and up to seven domains. The Plasmodium genome encodes around 70 PfEMP1 variants. To aid in structure based drug-design and vaccine development detailed structural knowledge for these proteins is required.
During the time course of this PhD thesis I have studied a number of PfEMP1 proteins using the SAXS technique. This biophysical technique has gained widespread attention during the past decade due to scientific and computational developments. SAXS is X-ray scattering from solutions and has in recent years proven very effective for obtaining structural information in the 10-15 Å resolution range for proteins. The technique is especially valuable for proteins that have proven difficult to crystallize for macromolecular high-resolution (<3 Å) crystallographic purposes. Macromolecular crystallography is typically the biophysical method of choice for obtaining detailed structural information but it unfortunately requires the formation of X-ray scattering protein crystals. The protein crystallization step remains a major bottleneck for X-ray protein crystallography. While conducting the SAXS experiments on PfEMP1 protein solutions, I alongside performed crystallization experiments using these solutions and found that the purified proteins showed very poor aptitude towards crystals formation. On the other hand, the SAXS method proved itself as a valuable tool for low-resolution structural characterization of the PfEMP1 proteins.
This thesis describes how small angle X-ray scattering (SAXS) was used to achieve low-resolution structural information for a range of PfEMP1 proteins investigated as potent malaria antigens. Especially the leading vaccine candidate against placental malaria, VAR2CSA was studied extensively. By the use of SAXS on multiple truncated VAR2CSA constructs it was possible to combine the acquired information and present a structural model for the DBL and CIDR domain arrangement in VAR2CSA. This work was published in Journal of Biological Chemistry (App I) [1]. VAR2CSA binds specifically to CSA in the placental tissue of pregnant women hereby causing severe malaria symptoms endangering both mother and child. The minimal VAR2CSA region required to effectively bind CSA was determined to be the N-terminal DBL domain, DBL2X which we locate in the SAXS derived VAR2CSA model next to DBL4, a DBL domain found to raise antibodies inhibiting CSA binding. Today, these domains of only 42 kDa each are further investigated as putative vaccine candidates against PAM.
Also I obtained the SAXS structures for a number of PfEMP1 proteins here among three of the ICAM-1 (Intercellular adhesion molecule 1) binding Type-A subtype PfEMP1s which are characterized by having an N-terminal conserved three-domain (DBL1α-CIDR1α-DBL2β) Head-structure carrying the ICAM-1 binding site. ICAM-1 is an immunoglobulin superfamily glycoprotein typically expressed on endothelial cell surfaces predominantly in the cerebral tissue. The PfEMP1s that bind ICAM-1 are the predominant cause of infected erythrocyte sequestration in the brain and are thus investigated as vaccine candidates against cerebral malaria. We found that two of these proteins, VAR13 [2], VAR20 together with a recombinant Head-structure construct (DC5), contrary to VAR2CSA, all showed elongated structures with domains assembled in a beads-on-a string manner.
Finally, in relation to this work, I also conducted SAXS measurements on the full-length five-Immunoglobulin-like domain ICAM-1 receptor protein. This is the first full-length structural description of this important receptor, which I show exists as a monomeric highly elongated structure in its solution state. The SAXS data indicates extensive conformational flexibility in the ICAM-1 molecule.
All in all, the combined contribution presented in this report covers many aspects of PfEMP1 structural composition and mechanism. Information that could be of value to future vaccine developments which ultimately will prevent the severe and fatal outcomes that malaria afflicts.
During the time course of this PhD thesis I have studied a number of PfEMP1 proteins using the SAXS technique. This biophysical technique has gained widespread attention during the past decade due to scientific and computational developments. SAXS is X-ray scattering from solutions and has in recent years proven very effective for obtaining structural information in the 10-15 Å resolution range for proteins. The technique is especially valuable for proteins that have proven difficult to crystallize for macromolecular high-resolution (<3 Å) crystallographic purposes. Macromolecular crystallography is typically the biophysical method of choice for obtaining detailed structural information but it unfortunately requires the formation of X-ray scattering protein crystals. The protein crystallization step remains a major bottleneck for X-ray protein crystallography. While conducting the SAXS experiments on PfEMP1 protein solutions, I alongside performed crystallization experiments using these solutions and found that the purified proteins showed very poor aptitude towards crystals formation. On the other hand, the SAXS method proved itself as a valuable tool for low-resolution structural characterization of the PfEMP1 proteins.
This thesis describes how small angle X-ray scattering (SAXS) was used to achieve low-resolution structural information for a range of PfEMP1 proteins investigated as potent malaria antigens. Especially the leading vaccine candidate against placental malaria, VAR2CSA was studied extensively. By the use of SAXS on multiple truncated VAR2CSA constructs it was possible to combine the acquired information and present a structural model for the DBL and CIDR domain arrangement in VAR2CSA. This work was published in Journal of Biological Chemistry (App I) [1]. VAR2CSA binds specifically to CSA in the placental tissue of pregnant women hereby causing severe malaria symptoms endangering both mother and child. The minimal VAR2CSA region required to effectively bind CSA was determined to be the N-terminal DBL domain, DBL2X which we locate in the SAXS derived VAR2CSA model next to DBL4, a DBL domain found to raise antibodies inhibiting CSA binding. Today, these domains of only 42 kDa each are further investigated as putative vaccine candidates against PAM.
Also I obtained the SAXS structures for a number of PfEMP1 proteins here among three of the ICAM-1 (Intercellular adhesion molecule 1) binding Type-A subtype PfEMP1s which are characterized by having an N-terminal conserved three-domain (DBL1α-CIDR1α-DBL2β) Head-structure carrying the ICAM-1 binding site. ICAM-1 is an immunoglobulin superfamily glycoprotein typically expressed on endothelial cell surfaces predominantly in the cerebral tissue. The PfEMP1s that bind ICAM-1 are the predominant cause of infected erythrocyte sequestration in the brain and are thus investigated as vaccine candidates against cerebral malaria. We found that two of these proteins, VAR13 [2], VAR20 together with a recombinant Head-structure construct (DC5), contrary to VAR2CSA, all showed elongated structures with domains assembled in a beads-on-a string manner.
Finally, in relation to this work, I also conducted SAXS measurements on the full-length five-Immunoglobulin-like domain ICAM-1 receptor protein. This is the first full-length structural description of this important receptor, which I show exists as a monomeric highly elongated structure in its solution state. The SAXS data indicates extensive conformational flexibility in the ICAM-1 molecule.
All in all, the combined contribution presented in this report covers many aspects of PfEMP1 structural composition and mechanism. Information that could be of value to future vaccine developments which ultimately will prevent the severe and fatal outcomes that malaria afflicts.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Chemistry, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 225 |
| Status | Udgivet - 2014 |
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