The chitinase-like proteins (CLPs) have evolved from chitinases, which degrade chitin, as part of a defence against microorganisms. However, the CLPs have lost the catalytic capability to degrade chitin. Instead, they have been associated with inflammatory processes and lung tissue remodelling during the type 2 immune response and during lung repair following a helminth infection. The molecular mechanisms underpinning these activities are not fully understood, but some CLPS are known to bind to heparin. We hypothesised that the function of CLPs may involve the interaction between CLPs and glycosaminoglycans (GAGs) in tissues. Here, the interactions of the highly related murine CLPs Ym1 and Ym2, and human YKL-40 were investigated. Using E. coli expression systems recombinant Ym1 and Ym2 were successfully expressed in SHuffle T7 lysY and BL21(DE3) plyS cells, respectively. A combination of chromatography techniques including anion exchange and size exclusion chromatography were used to purify both Ym1 and Ym2. SDS-PAGE analysis and electrospray ionisation mass spectrometry determined that the protein was homogeneous and had the expected molecular weight. Heparin affinity chromatography (HAC) and bio-layer interferometry (BLI) were used to investigate the GAG binding properties of Ym1 and Ym2, with more limited analysis of YKL-40, that was available commercially. We found that Ym1 and Ym2 interact with heparin and heparan sulphate (HS). Albeit with Ym2 only interacting at low pH and low salt strength (pH 5.5, 40 mM NaCl), indicating vestigial activity. Ym1 interacted with heparin/HS in a pH dependent manner at physiologically relevant salt strengths. When investigating the specificity of the heparin/HS interaction it was found that N-sulphation was key for the binding of both Ym1 and Ym2. Amino acids in Ym1 and Ym2 that may mediate heparin/HS binding were identified using analysis of sequence information combined with the available crystal structures of YKL-40/chitin and Ym1 (apo). These residues were either within or close to the 'chitin-binding' groove and site-directed mutagenesis was used to generate mutant CLPs, including the K80A_K198A and K80E_K198E mutants for both proteins. Characterisation of these (and other) mutants by HAC and BLI revealed they had reduced heparin/HS binding capabilities and indicated that the 'chitin-binding' groove was the heparin/HS binding site in Ym1 and Ym2. The higher affinity binding for heparin/HS seen with an acidic pH has revealed a potential mechanism for regulation of CLP function in vivo, through changes in tissue pH, for example during inflammation. The successful generation of CLPs mutants with reduced heparin/HS binding has provided reagents for future work to better understand the role of heparin/HS binding for CLPs function during tissue repair e.g., these can be used with Ym1 deficient mice in models of angiogenesis, asthma/allergen or helminth infection.
|Date of Award||1 Aug 2023|
- The University of Manchester
|Supervisor||Judi Allen (Supervisor), Anthony Day (Supervisor) & David Leys (Supervisor)|