Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species.
C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.
Boshra
Hani
H
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Li
Jun
J
Peters
Rodney
R
Hansen
John
J
Matlapudi
Anjan
A
Sunyer
J Oriol
JO
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002464:Cell Migration Inhibition; D002634:Chemotaxis, Leukocyte / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000302:isolation & purification; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D007962:Leukocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007106:Immune Sera; D044087:Receptor, Anaphylatoxin C5a