title: Functional implications of the Knops blood group polymorphisms on complement receptor 1 binding
Creators: name: Erdemci-Evin, Sümeyye
Publisher: name: The University of Edinburgh
Year: 2020
Subjects: subject: Complement receptor 1 (CR1/CD35)
subject: transmembrane protein
subject: complement pathway regulator
subject: Knops blood group
subject: Swain Langley 2 (Sl2) polymorphism
subject: McCoy b (McCb) polymorphism
subject: Long Homologous Repeat D (LHRD)
subject: Mannose Binding Lectin

description: Complement receptor 1 (CR1/CD35) is a transmembrane protein and complement pathway regulator. CR1 has been implicated in the pathogenesis of various infectious diseases through altering complement regulation or opsonising pathogensto facilitate their entry into host cells. There are a number of polymorphisms of CR1 which include copy number, size variants and single nucleotide polymorphisms (SNPs) in the gene sequence. One example of these polymorphisms is the Knops blood group, which includes the antithetical pairs: Swain-Langley1/2 and McCoy a/b. There is epidemiological evidence to suggest these polymorphisms may protect against infectious diseases including malaria. Recent analysis of a large case-control study of Kenyan children demonstrated that the Swain Langley 2 (Sl2) polymorphism in CR1 was associated with significant protection against cerebral malaria, whereas the McCoy b (McCb) polymorphism was associated with increased odds of cerebral malaria. The mechanism of these associations is still not well-defined, and this thesis aims to investigate how these polymorphisms may influence the pathogenesis of cerebral malaria through their binding of complement components. The Sl and McC polymorphisms are located in Complement Control Proteins (CCPs) 24-25 in Long Homologous Repeat D (LHRD) region of CR1 which also has binding sites for the initiators of lectin and classical pathways Mannose Binding Lectin (MBL) and C1q. I hypothesized that Sl2 and McCb polymorphisms may affect the risk of cerebral malaria through changing the affinity of MBL or C1q binding to CR1. To test this, I produced various short recombinant CR1 proteins containing CCPs 22-30 with different Sland McC mutations and expressed them in mammalian cells and observed their interactions with MBL and C1q using real time binding assays. I used previously produced pCRII plasmids containing CCPs 22-30 of CR1 into which various Sl and McC mutations had been introduced using site-directed mutagenesis. I then used the restriction enzyme BsMBI to cut the sequence of interest and ligate it into an expression vector to create pcDNA3.1(-)CCP22-30His. Final plasmids were created containing Sl1/McCa (wildtype), Sl1/McCb, Sl2/McCa, Sl2/McCb and rs4844609 (a nearby SNP in the LHRD region of CR1associated with Alzheimer’s disease risk). Successful clones were confirmed by Sanger sequencing and transfected into mammalian cells (293F cells). Following protein expression, cell culture supernatants were dialysedandproteins purified using nickel-nitrilotriacetic acid (Ni-NTA) columns. Protein characterization was performed by SDS-PAGE and western blotting with anti-CR1 monoclonal antibody E11 and human sera containing antibodies against McCa, McCb and Sl1 which confirmed that presence of expected Knops antigens. Surface plasmon resonance (SPR) was then performed to assess binding of the recombinant proteins to MBL and C1q. Surprisingly, SPR showed no binding of the wild type (Sl1/McCa) CCP22-30 recombinant protein to MBL or C1q. However, I also found no binding between MBL or C1q with commercial full length soluble CR1 (sCR1), in contrast to what would be expected from published reports. Therefore, my experiments were unable to confirm previous research showing that CR1 binds MBL and C1q. Contacting the authors of these original studies revealed that they had used some unorthodox methods in their SPR experiments.I then performed supplementary experiments to assess whether CR1 on erythrocytes bound to C1q protein spots in a Petri dish. I found that C1q only bound to CR1 under low ionic strength conditions and not under isotonic conditions. This suggests that the binding of C1q to CR1 is very weak. In conclusion, although my initial aim was to test whether the Knops blood group polymorphisms Sl and McC affect C1q or MBL binding to CR1, I was unable to test this hypothesis because,contrary to published data, my experiments showed no binding of the wild type protein. These results indicate the need for further research to determine whether MBL and C1q binding to CR1 does occur under physiological conditions.