No description available.

No description available.

Factor H (FH) is a 155-kDa plasma protein that regulates the alternative pathway of the complement system. Its 20 CCP modules, of 51-62 amino acid residues each, are linked by short stretches (“linkers’) of three to eight residues. We set out to test the hypothesis that long linkers towards the middle of FH play a role in ensuring that its architecture allows binding sites near its N- and C-termini to engage cooperatively with the main target, C3b, which is the key complement pathway-triggering product of C3 cleavage. In initial work, site-directed mutagenesis was used to test whether two mutations, R53H and R78G, located within CCP 1 and linked to the kidney disease atypical hemolytic uremic syndrome, are functionally deficient. Mutant versions and a native-sequence version of CCPs 1-4 of FH (i.e. FH 1-4) were tested for their ability to act as a cofactor for the FI-mediated cleavage of C3b, and accelerate the decay of the C3 convertase. It was shown that FH 1-4 R53H binds normally to C3b but has no regulatory activity while FH 1-4 R78G binds very poorly and is also deficient in cofactor and decay-accelerating activities. In subsequent work, mutagenesis was used to make the eight-residue CCPs 12-13 linker shorter (SL), or more flexible through introduction of glycine residues (3xGLY), within recombinant (r) module pair FH 12-13, and in rFH 10-15 and rFH 8-15 as well as full-length rFH. NMR showed CCPs 12 and 13 remain intact following mutation of the linker but (in FH 12-13) are more flexibly mutually disposed, as expected. SAXS indicated that both FH 10-15 SL and FH 10-15 3xGLY nonetheless have similar compact structures to native sequence (WT) FH 10-15. On the other hand, FH linker mutants interact with C3b (according to surface plasmon resonance) somewhat less well than WT FH and in the case of FH SL, affinity is similar to that of FH 19-20, i.e. there is no evidence that both C3b-binding sites in this mutant bind to the target simultaneously. Nonetheless, the bacterial protein PspCN boosts binding of linker mutants to C3b by a similar factor (three-to-fivefold) to that observed for FH WT. Thus, while interactions between non-sequential CCPs are important for FH architecture, a bend at the 12-13 linker is needed for full-length FH to adopt a fully biological activity confirmation. The use of EPR for structural studies of rFH and its mutants was explored. Free cysteines were engineered in so they could have spin labels site-specifically attached. Alternatively, a recognition site for transglutaminase was introduced so a spin label could be incorporated. These strategies were applied to rFH 12-13 and rFH 10-15 as a prelude to studies of full-length FH. Several suitably engineered proteins were prepared but only one paramagnetically labeled sample (of FH 12-13) made it for EPR; this yielded results commensurate with the NMR-derived structure. Taken together, these promising data lay the groundwork for a future, potentially very insightful, combined mutagenesis and EPR study of FH architecture and its role in complement activation.

No description available.

No description available.

NMR studies have long been used as a tool to derive structural and dynamic information. Such information has a wide range of applications, and notably is used in the study of structure-activity relationships. The aims of this work were to use NMR spectroscopy to derive structures of the molecules inhibiting the activation of the alternative pathway of the complement portion of the innate immune system (namely, the N-terminus of factor H (FH) and two small peptides, Compstatin 10 and Compstatin 20) and to consider the interdomain dynamics of proteins consisting of three modules theoretically (in silico) and experimentally (for the three N-terminal domains of FH). We focused on the three N-terminal complement control protein (CCP) domains of the important complement regulator, human factor H (i.e. FH1-3). Its three-dimensional solution structure was derived based on nuclear Overhauser effects and residual dipolar couplings (RDCs). Each of the three CCP modules in this structure was similar to the corresponding CCP in the previously derived C3b-bound structure of FH1-4, but the relative orientations of the domains were different. These orientations were additionally different from the interdomain orientations in other molecules that interact with C3b, such as DAF2-4 and CR1-15-17. The measured RDC datasets, collected under three different conditions in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids), were used to estimate interdomain motions in FH1-3. A method in which the data was fitted to a structural ensemble was used to analyze such interdomain flexibility. More than 80% of the conformers of this predominantly extended three-domain molecule exhibit flexions of < 40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring, as well as eventual reorganization of modules into the conformation captured in the previously solved crystal structure of a C3b complex with FH1-4. The NMR study of the Compstatin analogues revealed unique structural features that had not before been observed in this group of peptides. These features included two b-turns per peptide, neither of which was located in the ‘canonical’ regions in which b-turns were observed in previous molecular dynamics and NMR studies. The structures of Compstatin 10 and Compstatin 20 derived here were consistent with the isothermal calorimetry (ITC) and surface plasmon resonance (SPR) data recorded previously. In the in silico study of interdomain motion of three-domain proteins carried out here, the domains were represented as vectors attached to one another in a linear fashion. They were allowed to undergo Brownian motion biased by the potentials between the sequential vectors. The resulting trajectories were analyzed using model-free and extended model-free formalism. The degree of coupling of the interdomain motion with overall motion was determined, along with a representation of the overall motion. The similarity between the trajectories of the vectors transformed to this overall motion frame and the results obtained from the model-free analysis was determined.

Factor H (FH) is a key regulator of the complement system, the principal molecular component of innate immunity in humans. The tight regulation of the alternative pathway (AP) of complement by FH occurs on host cells as well as in fluid phase. FH regulation of AP is achieved through its C3b. Bb-decay accelerating activity and cofactor activity for C3b proteolysis by factor I. This study presents evidence that the first three CCP modules, i.e. FH~1-3, constitute the minimal unit with cofactor activity for factor I. The work presented in this thesis describes the recombinant protein expression and NMR-derived structure determination of two overlapping pairs, FH~1-2 and FH~2-3, together with the use of these structures to build a model of the FH~1-3 structure. A structural comparison with other C3bengaging proteins (namely factor B, complement receptor type 1 and decay accelerating factor) is presented and used to devise hypotheses as to the respective roles of the three modules during an encounter with the convertase. This thesis further describes an investigation of the structural effects of two disease-associated sequence variants in the context of FH~1-2: namely the single nucleotide polymorphism V62I linked to age-related macular degeneration, and the R53H mutation linked to atypical haemolytic uraemic syndrome.

Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

Glycoengineering is a technology that could improve protein therapeutics. While protein glycosylation in general enhances solubility and stability, and reduces aggregation, immunogenicity and proteolysis, specific kinds of glycosylation may also be critical. For example, capping of glycans with N-acetylneuraminic acid (Neu5Ac) maximises circulatory half-life in humans. Moreover, some glycans directly participate in molecular recognition and other aspects of glycoprotein function. Glycoproteins produced by non-human mammalian cells carry glycans capped by N-glycolyl-neuraminic acid rather than Neu5Ac. Yet production in human cell lines is costly and slow, requires specialist facilities, produces low yields and is subject to additional regulations. Hence there is a case for glycoengineering alternative expression systems capable of rapid, low-cost, high-yield glycoprotein production. This report focuses on the glycoengineering of Pichia pastoris, a yeast, to produce recombinant human glycoprotein factor H (FH) bearing human-like glycans. FH is a potent down-regulator of the complement system. Mutations and SNPs in FH result in autoimmune diseases such as atypical haemolytic ureamic syndrome and age-related macular degeneration (AMD). Recombinant FH is an enticing therapeutic candidate for treating AMD, but high doses are required since FH is abundant (200-300 mg l-1) in normal human serum. Human FH (155 kDa), with eight sites of N-linked glycosylation and 40 disulphides, is a challenging target for recombinant production. Yet FH was previously expressed to 10s of milligrams in P. pastoris. In this study, methods were established to confirm that human plasma-derived (h)FH carries predominantly N-linked diantennary disialylated complex-type glycans, with monosialylated diantennary structures and triantennary structures in fucosylated and non-fucosylated forms, contributing to glycan heterogeneity. Functional comparison of native hFH, enzymatically desialylated (DeSia-) hFH and deglycosylated recombinant P. pastoris-produced (DeGly-r)FH showed that DeSia-hFH had the lowest affinity for complement protein C3b, its key target. Moreover, DeSia-hFH binds C3d, an opsonic C3b-breakdown product, whereas native hFH does not. DeSia-hFH had an improved ability to accelerate decay of the C3 convertase (an enzyme that cleaves C3 to C3b) compared to native hFH, but neither was as good as DeGly-rFH in this respect. In contrast, DeGly-rFH had reduced cofactor activity (for factor I-mediated degradation of C3b) compared to native hFH whereas DeSiahFH did not have reduced cofactor activity. These data suggest that sialylation of FH glycans may play a role in stabilising a conformation of circulating FH that is not fully effective, consistent with specificity for self-surfaces and resistance to bacterial hijack. Aiming eventually to produce human-like glycosylated FH in glycoengineered P. pastoris, the SuperMan 5 strain served as a starting point. While conventional strains of P. pastoris put hypermannosylated N-linked glycans on proteins, glycans on SuperMan 5-produced FH were shown to contain just five mannose (Man) residues. In further glycoengineering, and following unsuccessful efforts to use inABLE technology for this purpose, commercially available (GlycoSwitch) vectors were used to introduce genes encoding the glycosyltransferase enzymes N-acetylglucosamine (GlcNAc) transferase I (GnTI) and galactose (Gal) transferase. These catalysed the formation of a hybrid-type glycan containing an N-acetyllactosamine (Gal-β(1,4)-GlcNAc (LacNAc)) antennae on a five-mannose glycan. Then two more GlycoSwitch plasmids, containing genes encoding α-Mannosidase II (ManII) and GnTII, were introduced into P. pastoris to catalyse the formation of a second LacNAc antennae. MALDI-TOF analysis found the glycosylation of this strain to be heterogeneous, containing the humanised diantennary digalactosyl glycan as well as other endogenous yeast glycans. This strain was designated SuperGal. Large-scale expression of rFH with terminally galactosylated complex-type glycans (Gal-rFH) in SuperGal yielded 100s of milligrams of purified Gal-rFH. Yeast-type glycans were enzymatically removed from rFH and the remaining complex-type humanised glycans were sialylated with a recombinant bacterial α(2,6)-sialyltransferase from Photobacterium sp. expressed in E.coli. Purified sialylated (Sia-) and non-sialylated (Gal-) rFH expressed in SuperGal were functionally characterised in vitro using SPR-based assays. In C3b-binding assays Sia-rFH had lower affinity compared to Gal-rFH. Both bound with lower affinity than DeGly-rFH. A similar pattern of binding affinity was seen for C3d. In C3 convertase decay-acceleration assays, all rFH glycoforms performed equally well and had greater activity than hFH. Conversely, Sia-and Gal-rFH were shown to perform equally as well as hFH in CA assays, while all three versions outperformed DeGly-rFH. However, in vivo complement activity assay carried out in a FH-knockout mouse model showed that humanisation of the glycosylation of rFH did not significantly improve activity compared to DeGly-rFH. In addition, analysis of the circulatory half-life of rFH showed that humanisation did not improve half-life. Further engineering steps will be required to increase the complex-type glycan site occupancy on rFH with a view to improving circulatory half-life and efficacy. However, this study represents a significant step forward in developing a therapeutically useful source of rFH.

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 127-137. Available online via the Internet.