Two Different Complement C3 Genes in Crocodilians
Crocodilians exhibit powerful antibacterial activities in their tissues and blood. The activities have been partially attributed to the presence of a potent serum complement system of proteins that acts in a nonspecific manner to kill bacteria. Complement activation involves activation of complement C3, a component with broad immune and regulatory function. We searched the crocodilian genomes (Alligator mississippiensis, Crocodylus porosus, and Gavialis gangeticus) for complement C3, and found two genes that code for isoforms with quite different sequences. Birds and mammals express only a single isoform of the complement C3 protein. Some snakes were shown to have two C3 genes; however, these encode proteins with very similar amino acid sequences. To date, only fishes were reported to express complement C3 isoforms with diversity similar to that of the crocodilian forms. Phylogenetic analysis suggests the gene duplication leading to the two crocodilian paralogs probably occurred within the order Crocodylia. Both contain signal sequences, putative internal thioesters, potential N-glycosylation sites, and functional domains that would allow them to interact with complement receptors and other complement system components. As has been suggested with fishes, the expression of multiple functional C3 isoforms may allow crocodilians to respond to a broad spectrum of immunological insult.
Crocodilian C3-1 and C3-2 nucleotide sequence identity values with other vertebrate species. Nucleotide sequence alignments were conducted using Se-Al (Rambaut, 2002) as described in the Materials and Methods. The results represent the means±standard deviations for each comparison. Human and chicken sequences were obtained from GenBank (Human, Homo sapiens NM_000064; Chicken, Gallus gallus NM_205405.1).
Amino acid identities of crocodilian complement C3-1 and C3-2 proteins with C3 from other vertebrates. Amino acid alignment and identities were performed using the electronic Genestream amino acid alignment tool as described in the Materials and Methods. The results represent the means±standard deviations for each comparison. Taxa and accession numbers are as follows: Mammals—Human, Homo sapiens NM_000064; Mouse, Mus musculus NM_009778; Killer whale, Orcinus orca XM_004277276; Domestic cow, Bos taurus NM_001040469; Pig, Sus scrofa NM_214009; Birds—Ground tit, Pseudopopodoces humilis XM_005533547; Chicken, Gallus gallus NM_205405.1; Pigeon, Columba livia XM_005513736; Falcon, Falco cherrug XM_005435629; Sparrow, Zonotricia albicollis XM_005496850; Squamates—Green Anole, Anolis carolinensis derived from genomic scaffold GAFN01002430; Cobra venom factor, Naja kaouthia U09969.2; Cobra C3, Naja naja L02365; Amphibian—African Clawed Frog, Xenopus tropicalis XM_002940048; Fishes—Rainbow Trout, Oncorhynchus mykiss L24433.1; Zebrafish, Danio rerio AB235997; Medaka, Oryzias latipes NM_001105083.
Phylogenetic analysis using parsimony of the derived amino acid sequences using PAUP v4.0a147. For the amino acid sequence matrix there were 1682 total characters with 357 constant characters and 1068 parsimony-informative characters. Taxa and accession numbers not given in Figure 2 are as follows: Painted turtle, Chrysemys picta XM_005310157.1; Coelacanth, Latimeria chalumnae XM_005994328.1.
Functional domains of the C3-1 and C3-2 proteins. For each functional region, identical aa are shaded dark, while aa substitutions with similar chemistries are shaded light. (A) Internal thioester region—the core region is highlighted in bold print, with the thioester formed between the thiol of the cysteine and the amide group of the glutamine. (B) N-terminal signal peptide—eukaryotic signal sequences are not well conserved at the level of sequence, but generally contain a stretch of hydrophobic aa that end with positively charged and polar aa at the C-terminal end. (C) Tetra-arginine linker at the junction of the C3a and C3b portions of the protein. (D) Properdin binding site—properdin binds to C3b and stabilizes its interaction with Factor B. (E) CR1, CR2, CR3, H, B binding region—upon binding to C3b, these receptors mediate immune complex clearance, phagocytosis, B-cell proliferation, alternative pathway activation, NO synthesis, degranulation, and other immunomodulatory effects. (F) The C3aR binding site—binding of C3a to C3aR results in mediation of a variety of anaphylatoxin activities.
Contributor Notes
Associate Editor: D. S. Siegel.