Investigating the Oligomerization of Vitronectin

Vitronectin is a multi-functional glycoprotein that is present in the plasma and extra-cellular matrix of eukaryotes. It is capable of binding a wide variety of structurally different ligands, including plasminogen activators, plasminogen activator-inhibitors, proteases, cell surface receptors and components of the extra-cellular matrix. Vitronectin exists in two conformations – as a monomer in circulation, and as a multimer in the extracellular matrix. The pathway by which vitronectin undergoes the transition from monomer to multimer is not well characterized, but this laboratory has put forward evidence to suggest that the binding of vitronectin with plasminogen activator inhibitor type-1 (PAI-1), facilitates higher order complex formation. Because multimeric vitronectin remains long after PAI-1 has dissociated from the complex, the question of which region(s) within vitronectin are interacting to maintain the multimeric conformation has been asked. To address this question, candidate domain regions in vitronectin were analyzed for potential aggregative properties. Based on this analysis, the Central domain of the protein was hypothesized to be the region responsible for self-association. In support of this hypothesis, the predicted structure of the Central domain is a 4-bladed β–propeller fold, a motif that is capable of mediating protein-protein interactions. However, not all β–propeller proteins have self-associative behavior. Examples include hemopexin, gelatinase-A and collagenase-3, all of which share distant sequence homology with vitronectin. In this study, a negative-design approach was used in which the Central domain of vitronectin was replaced with the homologous β–propeller regions from hemopexin and gelatinase-A. The substitution of this domain with β–propeller regions that do not demonstrate self-association was expected to alter the aggregative properties of the chimeras. An additional recombinant form of vitronectin was also designed which did not possess the C-terminal domain to conclusively rule out this domain as the candidate region. In addition to this approach, limited proteolysis was used to try and isolate the Central domain region of vitronectin so that it could be better characterized both structurally and functionally. All three recombinant proteins were synthesized and expressed in insect Sf9 cells. However, purification of these proteins was met with difficulty due to the low expression levels observed. Optimization attempts to improve expression were unsuccessful, and so structural and functional analysis studies were limited. Nevertheless, all three proteins were able to bind to wild-type PAI-1 in solution, as well as demonstrate Western blot reactivity with two monoclonal antibodies. Initial results from the limited proteolysis study also showed potential peptide fragments for the Central domain of vitronectin. Based on the results from this study, the region responsible for the selfassociation of vitronectin should be further examined and resolved to critical residues within the Central domain. These results may offer insight into the process of oligomerization in other proteins, many of which are implicated in fatal diseases, e.g. Alzheimer’s and Creutzfeldt-Jakob disease.