Purification, characterization, and amino acid sequence of rat anaphylatoxin (C3a)

Abstract

C3a anaphylatoxin derived from the third component of complement has been isolated from rat serum and its complete amino acid seuqence determined. A three-step purification procedure was employed that consisted of gel filtration on Sephadex G-100, followed by chromatography of the anaphylatoxin-containing pool on carboxymethylcellulose. A subsequent separation on DEAE-Sephadex resolved C3a from minor contaminating peptides. Biological studies have shown that purified rat anaphylatoxin is approximately twice as active as human or porcine C3a when tested for smooth-muscle contraction. In addition to the active form of rat anaphylatoxin, a serum carboxypeptidase B inactivated form of C3a (C3ades-Arg) was purified from rat serum and utilized in subsequent structural studies. Sequence analysis of rat C3a was facilitated by a long automated Edman degradation which established the first 55 residues of the anaphylatoxin. Overlapping peptides were generated by cyanogen bromide and trypsin, and the resultant fragments were sequenced by either automated or manual Edman procedures. The primary structure of rat C3a is 70% identical to the previously determined structures of human and porcine anaphylatoxin. Antisera raised to the purified rat peptide do not cross-react immunologically by Ouchterlony analysis with either human or porcine C3a.

Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

“…Therefore, nucleotide sequences of a portion of this fragment were determined; the corresponding amino acid sequence of C3a and its flanking regions was derived (Fig. 4) and compared with the known C3a amino acid sequences of other species (29)(30)(31)). These results demonstrate that mouse C3a shares 72, 52, and 50 residues, respectively, out of 78 with rat and out of 77 with human and porcine C3a.…”

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

“…It is noteworthy that the disulfide bridges in human C3a appear to be unusually labile (Chang et al, 2008), a phenomenon that may provide additional regulation of the activity of C3a in vivo. Guinea pig C3a has 70% identity with rat (Jacobs et al, 1978), human, porcine (Corbin and Hugli, 1976), and mouse (Hugli et al, 1975b), and C3a from these species have identical activity in smooth muscle contraction, histamine release, and vascular permeability assays. Among all C3a sequences, the overall arrangement of basic residues is also highly conserved, with a ubiquitous C-terminal Arg.…”

Role of the Complement Anaphylatoxins in Inflammation and Hypersensitivity Reactions in the Lung

“…C3 and C5, respectively [19][20][21][22][23]. A similar peptide, C4a, derives analogously from C4 and has been described as possessing some anaphylatoxic properties as well [24].…”