Biochemical and functional studies of a novel complement inhibitor, CRIT, with its interaction partners

Abstract

Complement C2 receptor trispanning (CRIT), a three transmembrane receptor, was first discovered on the surface of the parasite Schistosoma haematobium and formerly termed Schistosoma trispanning orphan receptor (Sh-TOR). This receptor acts as decoy C2- binding receptor to protect the parasite from classical pathway-mediated complement attack by competing with C4 for the binding of C2. The first extracellular domain (ed1) proved to be the hot spot for the binding of C2. The first part of my work and the detailed results will be presented and discussed in Section I. Apart from Schistosoma, another parasite Trypanosoma cruzi and rat also express CRIT and show high sequence homology. The cloning of human CRIT was started by PCR with the templates from both genomic DNA and cDNA. The primers used were selected from the conserved region. The human CRIT nucleotide sequences from genomic DNA and cDNA are identical, implying that human CRIT is encoded by a single exon, and therefore an intronless gene. A phylogenetic analysis of the available CRIT genes giving a measure of their evolutionary distance shows that the parasite species are as far removed from their human host as is the rat sequence. This is thought to be an example of horizontal gene transfer (HGT) in terms of genetic transfer between host and parasite. In addition, the cellular distributions and localizations of human CRIT were determined by immonblotting and immunohistochemistry. It has been showed that a major binding site for C2 is located on short almost identical peptide sequences of CRIT-ed1 and the β-chain of C4. The C2 domain(s) involved in these bindings, however, has remained unknown. Since most of the von Willebrand factor-A (vWFA) domains are components of the extracellular matrix and very often are the sites for protein-protein interaction in cell adhesion protein, such as integrins, the vWFA domain of C2 was speculated to be the potential site of interaction with CRIT and C4. To further address this question, the vWFA domain (amino acids 224-437) of C2 was cloned and the first functional recombinant vWFA protein was expressed and purified. Based on the functional data, the vWFA domain of C2 is a potential binding site for both C4 and CRIT, and this forms the major aspect of Section II. The major findings of the regulatory effect of CRIT on the alternative pathway are summarized in Section III. In view of the fact that the classical and alternative complement pathways have many functional and structural similarities, the regulatory effect of CRIT on the alternative pathway was further investigated. In this study, as 11 amino acid peptide (CRIT-H17) derived from the C-terminal part of CRIT-ed1 was used, as it was found to be a potent inhibitor of the classical pathway. The data presented indicates that CRIT-H17 also functions as a regulator of the alternative pathway. Besides binding C2, CRIT-H17 was shown to bind factor B and its two fragments, Ba and Bb, and C3b but not factor D. For this, interacting proteins were stabilized with a crosslinking agent and complexes detected by immunoblots. CRIT-H17 bound to FB, possibly at the junction of Ba and Bb near the factor D cleavage site, and blocked the factor Dmediated cleavage of FB. Unlike DAF, CRIT-H17 had no decay accelerating activity on the C3bBb complex. Additional experiments showed that CRIT-H17 partially inhibited the factor I-mediated cleavage of C3b in the presence of factor H or CR1. These data indicate that the regulatory role of CRIT-H17 in the alternative pathway is complex, probably related to its dual binding to factor B and C3b.