Tokio Terado

Molecular Cloning of C4 Gene and Identification of the Class III Complement Region in the Shark MHC

To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.

Conservation of the modular structure of complement factor I through vertebrate evolution

Mammalian complement factor I plays pivotal roles in the regulation of complement activation and generation of important biological activities from C3. The evolutionary origin of factor I has been unclear except with regard to the molecular cloning of factor I from amphibian Xenopus. Here, we report the identification and characterization of factor I cDNA from the liver of the banded houndshark. The deduced amino acid sequence of shark factor I showed a modular organization that was completely identical to that of mammalian factor I, suggesting the functional conservation of factor I throughout vertebrate evolution. Functionally important amino acid residues such as the basic residues at the processing site and the residues at the active site of the serine protease domain are conserved. Repeated sequences composed of 16 amino acids were inserted at a site between the leader peptide and the factor I/membrane attacking complex module in the shark factor I. This repeat is missing from mammalian and amphibian factor I, and the biological significance of the sequence, if any, is not clear at the moment. There was only one copy of the shark factor I gene, and Northern blotting analysis showed that the shark factor I gene was expressed only in the liver among several organs tested. While the lack of functional data does not exclude the possibility that factor I could have a different function, all these facts, together with the earlier reported data suggest the existence of a well developed complement system in cartilaginous fish.

The enhancement of cell lethality and changes in production and repair of DNA damage by hypertonic treatment after exposure to X rays, bleomycin, and neocarzinostatin.

Chinese hamster ovary cells were treated with hypertonic 0.5 M NaCl solution after exposure to X rays or the radiomimetic drugs bleomycin or neocarzinostatin. The cytotoxicity of these agents was greatly enhanced by the hypertonic treatment. On the other hand, no effect was observed after exposure to ultraviolet light, and a significant effect was obtained with mitomycin C (MMC), adriamycin (ADR), and ethyl methanesulfonate (EMS). Assays by filter elution revealed that hypertonicity had various effects on the damage produced by the different agents. Strand breaks resulting from exposure to X rays and radiomimetic agents were sensitive to hypertonic treatment. Hypertonicity caused the production of new lesions and inhibited the rejoining of DNA strand breaks, both of which may be responsible for the enhanced cytotoxicity. On the other hand, the formation of crosslinks by MMC or protein-associated double-strand breaks by ADR, the major forms of damage by which these agents cause cytotoxicity, was not affected by hypertonic treatment. As strand breaks are known to be produced by MMC or ADR, they could account at least partly for the sensitization. However, various kinds of damage are produced by MMC, and any of these could be involved in the sensitization. To our knowledge EMS produces only base damage. Thus hypertonic treatment may have an effect on various types of damage.

Evolutionary analysis of two complement C4 genes: Ancient duplication and conservation during jawed vertebrate evolution

ABSTRACT The complement C4 is a thioester‐containing protein, and a histidine (H) residue catalyzes the cleavage of the thioester to allow covalent binding to carbohydrates on target cells. Some mammalian and teleost species possess an additional isotype where the catalytic H is replaced by an aspartic acid (D), which binds preferentially to proteins. We found the two C4 isotypes in many other jawed vertebrates, including sharks and birds/reptiles. Phylogenetic analysis suggested that C4 gene duplication occurred in the early days of the jawed vertebrate evolution. The D‐type C4 of bony fish except for mammals formed a cluster, termed D‐lineage. The D‐lineage genes were located in a syntenic region outside MHC, and evolved conservatively. Mammals lost the D‐lineage before speciation, but D‐type C4 was regenerated by recent gene duplication in some mammalian species or groups. Dual C4 molecules with different substrate specificities would have contributed to development of the antibody‐dependent classical pathway. HIGHLIGHTSTwo C4 isotypes were found in various jawed vertebrates, including sharks and birds/reptiles.C4 gene duplication occurred in the early days of the jawed vertebrate evolution.Two divergent C4 lineages, termed H‐lineage and D‐lineage, were found in bony vertebrates.The D‐lineage genes were located in a syntenic region outside MHC.In mammals, the D‐lineage was lost before speciation, but D‐type C4 was regenerated in some species or groups.