C2 by-pass: Cross-talk between the complement classical and alternative pathways.

Several disorders associated with the total or partial absence of components of the human complement system are known. Deficiencies of classical pathway (CP) components are generally linked to systemic lupus erythematosus (SLE) or SLE-like syndromes. However, only approximately one-third of patients who lack C2 show mild symptoms of SLE. The relatively high frequency of homozygous C2 deficiency without or with minor disease manifestation suggests that there might be a compensatory mechanism which allows the activation of the CP of complement without the absolute requirement of C2. In this study we show that factor B (FB), the C2 homologue of the alternative pathway (AP) of complement, can substitute for C2. This was confirmed by using C4b as immobilised ligand and FB as analyte in Surface Plasmon Resonance (BIACORE). C2 binding to the immobilised C3b-like molecule C3(CH3NH2) was not seen. The estimated binding constant for C4bB complex formation was 2.00 * 10-5 [M]. We were further able to demonstrate that C4b supports the cleavage of Factor B by Factor D. Finally, cleavage of 125I-C3 by C4bBb was evaluated and gave strong evidence that the "hybrid" convertase C4bBb can cleave and activate C3 in vitro. Cleavage activity is very low, but consistent with some of the "C2-bypass" observations of others.

CRIT is expressed on podocytes in normal human kidney and upregulated in membranous nephropathy.

Complement C2 receptor inhibitor trispanning (CRIT) is a novel human complement regulatory cell surface receptor. It binds the human complement protein C2 and blocks the classical pathway of complement activation, thus protecting the cell against complement attack. CRIT expression in the kidney was analyzed by immunohistochemistry and in situ hybridization. Normal kidney and renal biopsies of patients with different nephropathies were studied. In glomeruli, CRIT protein was expressed only in podocytes. CRIT was also detected in endothelial cells of arterioles and arteries, but not of veins and peritubular and glomerular capillaries. A homogeneous and marked upregulation of CRIT was observed in podocytes in membranous nephropathy (MN). In focal and segmental glomerulosclerosis (FSGS) and minimal change disease, CRIT was downregulated in glomeruli with a loss of the staining in sclerotic lesions of FSGS. No specific changes were observed in the other nephropathies studied. However, podocytes showed in all pathologies a redistribution of CRIT in the cell bodies of 'activated' podocytes. The intensity of mRNA transcription correlated directly with the protein staining in the normal kidney and in MN. These data indicate that CRIT is expressed in the normal human kidney essentially by glomerular podocytes and arterial endothelial cells. The podocyte CRIT expression is upregulated in MN, which is in strong contrast with the known loss of podocyte complement receptor 1. CRIT might represent the last line of defense against complement aggression in MN, and the upregulation of CRIT in 'activated' podocytes might represent a similar self-defense mechanism.

A Schistosoma protein, Sh-TOR, is a novel inhibitor of complement which binds human C2.

Human complement regulatory (also called inhibitory) proteins control misdirected attack of complement against autologous cells. Trypanosome and schistosome parasites which survive in the host vascular system also possess regulators of human complement. We have shown Sh-TOR, a protein with three predicted transmembrane domains, located on the Schistosoma parasite surface, to be a novel complement regulatory receptor. The N-terminal extracellular domain, Sh-TOR-ed1, binds the complement protein C2 from human serum and specifically interacts with the C2a fragment. As a result Sh-TOR-ed1 pre-incubated with C2 inhibits classical pathway (CP)-mediated haemolysis of sheep erythrocytes in a dose-dependent manner. In CP-mediated complement activation, C2 normally binds to C4b to form the CP C3 convertase and Sh-TOR-ed1 has short regions of sequence identity with a segment of human C4b. We propose the more appropriate name for TOR of CRIT (complement C2 receptor inhibitory trispanning).

Schistosoma TOR (trispanning orphan receptor), a novel, antigenic surface receptor of the blood-dwelling, Schistosoma parasite.

Sh-TOR is a novel, putative three transmembrane domain receptor of Schistosoma haematobium, which has no extensive homology to any other known protein. The 0.86 kb open reading frame was found to encode a novel protein, 286 amino acids long and of 32 kDa. It was shown that Sh-TOR can be phosphorylated on tyrosine and the protein sequence reveals a long cytoplasmic tail with several consensus phosphorylation sites for enzymes which characteristically associate with membrane receptors. The proposed topology of Sh-TOR, based on antibody recognition of transfected Sh-TOR, predicts that the amino terminus is extracellular and the carboxyl terminus intracellular. Sh-TOR is a non-glycosylated protein found in the surface tegumental plasma membrane, and tegumental surface pits of adult schistosomes. The 1.35 kb transcript was most highly expressed in the larval stage, which is more susceptible to immune attack. A TOR homologue from Schistosoma mansoni is also described. A homologue from Trypanosoma cruzi, another human parasite was also isolated, but not from the free-living nematode Caenorhabditis elegans. Recombinant Sh-TOR is specifically recognised by a passively protective serum, from baboons vaccinated with irradiated Schistosoma parasite. Together with its surface location, this means that Sh-TOR is also a potential vaccine candidate molecule.

phi 20, a temperate bacteriophage isolated from Bacillus anthracis exists as a plasmidial prophage.

This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage phi 20 isolated from B. anthracis Sterne 34F2 (pXO1+ pXO2-) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The phi 20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.