Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis.
The recent identification of complement components in deuterostome invertebrates has indicated the presence of a complement system operating via an alternative pathway in echinoderms and tunicates and via a MBL-mediated pathway thus far identified only in tunicates. Here, we report the isolation of two C3-like genes, CiC3-1 and CiC3-2, from blood cell total RNA of the ascidian Ciona intestinalis. The deduced amino acid sequences of both Ciona C3-like proteins exhibit a canonical processing site for alpha and beta chains, a thioester site with an associated catalytic histidine and a convertase cleavage site, thus showing an overall similarity to the other C3 molecules already characterized. Southern blotting analysis indicated that each gene is present as a single copy per haploid genome. In situ hybridization experiments showed that both CiC3-1 and CiC3-2 are expressed in one type of blood cell, the compartment cells. Two polyclonal antibodies, raised against two deduced peptide sequences in the alpha chain of CiC3-1 and CiC3-2, allowed the identification by Western blot of a single band in the blood serum, of about M(r)150,000. A phylogenetic tree, based on the alignment of CiC3-1 and CiC3-2 with molecules of the alpha(2)-macroglobulin superfamily, indicated that the Ciona C3s form a cluster with Halocynthia roretzi C3. The phylogenetic analysis also suggested that the duplication event from which the CiC3-1 and CiC3-2 genes originated occurred in the urochordate lineage after the separation of the Halocynthia and Ciona ancestor.
Marino
Rita
R
Laboratory of Cell Biology, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy.
Kimura
Yuko
Y
De Santis
Rosaria
R
Lambris
John D
JD
Pinto
Maria Rosaria
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002938:Ciona intestinalis / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D003176:Complement C3 / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D003176:Complement C3; D018076:DNA, Complementary