Structure-function analysis of collectins CL-Kl, CL-L1 and CL-LK in the innate immune system

The complement system is an essential part of the innate immune response, which is a protein cascade that interact with each other to form a network to give the body protection, it also stimulates a series of inflammatory responses. Complement is activated by three pathways: Classical (CP), Lectin (LP) and Alternative pathways (AP). The LP can be activated through recognition components (RMPs), like collectin kidney-1 (CL-K1) and CL-Liver (CL-L1), it has been reported that they form heterooligomers in serum, called CL-LK. These recognition molecules bind to the pathogen in Ca+2 dependant manner, and in association with three proteases (MASPs), however relatively little is known about their role in host defence or their sugar specificities. The aim of my project is to demonstrate structure-function analysis of CL-L1, CL-K1 and CL-KL with the goal of understanding how these collectins function within the innate immune system.

While CL-K1 has been crystallised in complex with mannose-containing ligands there are no structures bound to fucosylated ligands such as blood group antigens and Lewis antigen, which are likely to play a role in ischaemia reperfusion injury. In this project I have achieved 13 novel Structures of the CRD of CL-K1 bound to: Monosaccharides (D-galactose, N-acetyl-D-glucosamine D-glucose, as well as D-mannose and L- fucose) and fucosylated ligands ABO antigens (A, B, H, H type 1 and H type 2) and Lewis antigens (Lea, Leb and Ley), were solved. CL-K1 showed an interesting way of binding, it can binds to the ligands in both orientation of the fucose residue.That highlights the versatility of the CRD and explains in part its relatively broad specificity. Binding occurs to a shallow groove CRD of CL-K1, formed between a tyrosine and two arginine residues. In all of the structures a fucose interacts to the ligands via the primary binding site in the CRD with the Ca2+.

In addition I attempted to produce recombinant forms of CL-L1 and CL-LK. Whilst it was possible to produce the CRD of CL-L1, production of larger fragments were unsuccessful.