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dc.contributor.advisorHughes, Jeremy
dc.contributor.advisorRichards, Anna
dc.contributor.advisorBarlow, Paul
dc.contributor.authorKerr, Heather
dc.date.accessioned2022-09-21T12:40:49Z
dc.date.available2022-09-21T12:40:49Z
dc.date.issued2022-09-21
dc.identifier.urihttps://hdl.handle.net/1842/39367
dc.identifier.urihttp://dx.doi.org/10.7488/era/2617
dc.description.abstractComplement Factor H (FH) is a 155-kDa plasma glycoprotein that regulates the alternative pathway of the complement system. Mutations or deficiencies in FH are associated with some cases of the kidney diseases C3 glomerulopathy and atypical haemolytic uraemic syndrome (aHUS). Recombinant human FH could potentially be used in some patients as a therapy for these diseases, both of which confer significant morbidity. This requires animal trials as well as a better understanding of which patients are likely to benefit most from FH-supplementation therapy. An abundance of FH in the plasma of healthy individuals implies that high doses of any potential recombinant FH protein will be needed. Previously, Pichia pastoris containing codon-optimised DNA encoding human FH yielded useful but relatively small quantities of protein. In other experiments plasma-derived murine and human FH rapidly controlled C3 turnover in the murine model of C3 glomerulopathy, a FH-deficient mouse strain. Difficulties in obtaining large quantities of pure well characterised murine FH and the rapid development of an immune response to human FH prevented longer-term trials of these agents. This study aimed to generate recombinant murine FH (rmFH) in sufficient quantities for thorough functional and biophysical characterisation and subsequently in vivo studies in FH-deficient mice. It further aimed to enhance production of recombinant human FH to allow assessment of the functional implications of disease-linked mutations. The methodology that had yielded recombinant human FH in P. pastoris generated only minimal quantities of rmFH. Bearing in mind the 40 disulfides present in FH, a new host P. pastoris strain was developed containing an expression-optimised gene for protein disulphide isomerase (PDI). This produced multi-milligrams of rmFH per litre. The rmFH had an affinity for both human and murine C3b similar to native mFH and was functional in co-factor and decay acceleration complement assays. The rmFH was also functional in the FH-deficient mouse model – it increased plasma C3 levels and reduced glomerular C3 staining 24 hours after a single intra-peritoneal injection. The half-life of circulating rmFH was, however, short, and the mice developed antibodies against the recombinant protein after ten days of daily dosing. Despite this, a reduction in glomerular C3 was still seen at ten days implying rmFH may have persisted for longer in the glomerular basement membrane. Mutations in FH are implicated in aHUS, a life-threatening kidney disease. Recombinant human FH was made with variations in both the N (I62V, R53H) and C terminus (D1119G, S1191L V1197A), and tested in assays for decay-acceleration, co-factor and complement-mediated haemolysis of (self-like) sheep erythrocytes. The addition of PspCN, a bacterial binding protein, enhanced co-factor activity and haemolysis protection, and therefore may reverse the deficiencies of some FH mutant variants.en
dc.contributor.sponsorMedical Research Council (MRC)en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.relation.hasversionHerbert AP, Makou E, Chen ZA, Kerr H, Richards A, Rappsilber J, et al. Complement Evasion Mediated by Enhancement of Captured Factor H: Implications for Protection of Self-Surfaces from Complement. Journal of immunology (Baltimore, Md : 1950). 2015;195(10):4986-98en
dc.relation.hasversionKerr H, Wong E, Makou E, Yang Y, Marchbank K, Kavanagh D, et al. Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation. The Journal of biological chemistry. 2017;292(32):13345-60en
dc.relation.hasversionYang Y, Denton H, Davies OR, Smith-Jackson K, Kerr H, Herbert AP, et al. An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy. J Am Soc Nephrol. 2018;29(6):1649-61en
dc.subjectFactor Hen
dc.subjectFH proteinen
dc.subjectC3 glomerulopathyen
dc.titleRecombinant complement factor H in complement-mediated diseasesen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen
dc.rights.embargodate2023-09-21en
dcterms.accessRightsRestricted Accessen


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