C-reactive protein: A study of its functional domains using transgenic mice

C-reactive protein (CRP) is an acute phase protein in humans whose plasma concentration can increase 1,000x following an inflammatory stimulus. Two of the most widely studied properties of CRP are its ability to bind phosphocholine (PCh) and to activate complement. These in vitro properties of CRP have been hypothesized to play a critical role in CRP function. Initially, we generated transgenic mice expressing rabbit CRP (rbCRP) to develop an in vivo assay for rbCRP function. These animals have been used to demonstrate that transgenic rbCRP had a net anti-inflammatory effect in mouse models of inflammation which include models of endotoxemia, pulmonary alveolitis, and antigen induced arthritis.; In order to understand the mechanism through which rbCRP imparts its effects, we created additional lines of transgenic mice expressing variants of rbCRP. Based on the co-crystal structure of C-reactive protein and phosphocholine, a variant of rbCRP (F66Y/E81K) was created that was incapable of binding PCh. A second variant of rbCRP (Y175A) with an altered ability to activate complement was generated based on site-directed mutagenesis studies.; To test the importance of these two functional domains of rabbit CRP in vivo we utilized these mice in a model in which they were challenged with a lethal dose of a mediator of inflammation, platelet activating factor (PAF). Our results indicated mice expressing wild type and Y175A rbCRP were protected against challenge by PAF, while mice expressing F66Y/E81K rbCRP were not. PAF contains a PCH moiety, and in an in vitro solid phase binding assay, wild type and Y175A rbCRP could bind PAF, but F66Y/E81K rbCRP could not.; We conclude that PCh binding is critical for the rbCRP protective effect and hypothesize that the mechanism of rbCRP protection from PAF lethality involves sequestration of PAF by rbCRP so that PAF is unable to bind its receptor. We further conclude that its ability to activate complement is not necessary for protection from PAF challenges. These studies are the first to dissect the functional domains of rbCRP in an in vivo pathophysiological setting.