Generation, purification and functional characterization of three C3a anaphylatoxins in rainbow trout: Role in leukocyte chemotaxis and respiratory burst

Rotllant, Josep; Parra, David; Peters, Rodney; Boshra, Hani; Sunyer, J. Oriol

doi:10.1016/j.dci.2003.11.001

Cited by 40 publications

(28 citation statements)

References 35 publications

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“…Although in mammals C5a is the most potent chemoattractant anaphylatoxin, very little is known about the role of fish or other nonmammalian vertebrate anaphylatoxins in chemotaxis. Recent studies in our laboratory have shown that none of the three purified C3a anaphylatoxin molecules derived from the three trout C3 isoforms appears to play a role in the chemotaxis of trout leukocytes (70). However, we have shown that complementactivated trout serum and the gel filtration-purified chemoattractant fraction both elicit strong chemotactic responses (Fig.…”

Section: Inhibition Of Chemotaxis By Anti-tc5ar Absmentioning

confidence: 75%

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

Boshra

Peters

et al. 2004

The Journal of Immunology

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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.

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Section: Inhibition Of Chemotaxis By Anti-tc5ar Absmentioning

confidence: 75%

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

Boshra

Peters

et al. 2004

The Journal of Immunology

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“…Cell migration was applied using a 96-well chemotaxis chamber (ChemoTX system, Neuroprobe) as previously described [32,35] to assess the chemoattractant activities of recombinant C5a, G and GC5a (10 nM in DPBS, PBS with 100 mg/L CaCl 2 and 100 mg/L MgCl 2 , pH 7.4) to trout periphery blood leukocytes (PBLs). DPBS (pH 7.4) and complement-activated trout serum (diluted 1/1000 in DPBS, pH 7.4) were used as the negative and positive controls respectively.…”

Section: Cell Migration Analysismentioning

confidence: 99%

Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen

LaPatra

et al. 2014

Fish & Shellfish Immunology

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“…The band of cells lying at the interface was collected, and the cells were washed with HBSS or kept in EMEM supplemented with 100 U/ml penicillin and 100 g/ml streptomycin. HKLs were isolated as previously reported (39).…”

Section: Isolation Of Pbls and Head Kidney Leukocytes (Hkls)mentioning

confidence: 99%

“…A recent report in trout showed for the first time in teleosts the presence of three C3a molecules generated from three trout C3 isoforms (C3-1, C3-3, and C3-4). Each of the three C3a isoforms stimulated to a significant degree the respiratory burst of trout head kidney leukocytes, suggesting that teleost fish contain a C3aR (39). Recently, recombinant trout C5a has been produced and shown to play a prominent role in inducing leukocyte chemotaxis (40,41) and respiratory burst (40).…”

mentioning

confidence: 99%

Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a Receptors in Nonmammalian Species

Boshra

Wang

Hove‐Madsen

et al. 2005

The Journal of Immunology

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Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

show abstract

Generation, purification and functional characterization of three C3a anaphylatoxins in rainbow trout: Role in leukocyte chemotaxis and respiratory burst

Cited by 40 publications

References 35 publications

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

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

Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen

Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a Receptors in Nonmammalian Species

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