Primary structure and functional characterization of rat C5a: An anaphylatoxin with unusually high potency

Abstract

The anaphylatoxin C5a is a pro-inflammatory factor generated from C5 during complement activation. C5a derived from rat C5 exhibits significantly greater potency compared to C5a from other species. Rat C5a was 25-fold more potent than human C5a for eliciting spasmogenic contraction of guinea pig ileum. Proteolytic removal of the C-terminal arginine of C5a (CSadesArg) reduced spasmogenic potency of rat C5a by only 4-fold compared to a 3,000-fold reduction for human CSadesArg. In addition, rat C5qesArg was 50-fold more potent than human ChdesArg in a guinea pig vascular permeability (in vivo) assay and as a chemotactic factor for human neutrophils. C5a and C5qesArg were purified from zymosan-activated rat serum. Rat C5a, like human C5a, is glycosylated but contains 77 amino acid residues instead of the 74 residues of human C5a. Comparison of the primary structures of rat and human C5a indicated differences at 30 positions including an insert of 3 residues (LLH) in the rat molecule between residue positions 3 and 4 in human C5a. Insertion of residues LLH between Gln-3 and Lys-4 in a recombinant human C5a molecule using site-directed mutagenesis failed to enhance potency. Synthetic C-terminal analogues of rat C5a proved to be measurably more potent than the corresponding human C5a analogues (Ember JA et al., 1993, Protein Sci Z(Supp1 1):159 [Abstr]). We conclude that multiple sequence differences in the C-terminal effector portion and/or elsewhere in rat C5a, but not the LLH insert, account for the significant enhancement in potency of rat C5a over C5a from other species.Keywords: anaphylatoxin; complement; C5a; rat; spasmogen As early as 1910, Friedberger demonstrated an activity in complement-activated animal sera that when administered intravenously was lethally toxic to guinea pigs (Friedberger, 1910). Because the treated animals appeared to undergo an anaphylactoid shock reaction, the term anaphylatoxin was coined to identify the lethal substance (Friedberger, 1910). Later it was reported that C5a generated in C-activated rat serum exhibited unusually potent in vivo effects in guinea pigs in comparison to C5a derived from other species (Friedberger et al., 1964). In

Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co‐cultures

“…However, the recombinant rat C5a used in the current investigation was synthesized according to sequence data in the Swiss-Prot protein sequence data bank. It differed from the recently published sequence [26] in amino acid position 55 (K-> N), 63 (P-> H), 68 (Q-> E) and contained 12 additional N-terminal residues (see section 2). In addition the recombinant protein was not glycosylated.…”

“…However, in other biological systems native rat C5a elicited half maximal effects already at lower concentrations. Thus, the EDs0 in a leukocyte chemotaxis assay was about 0.01/lg/ml [26], and the EDs0 for the contraction of guinea pig ileum was even as low as 0.3 ng/ml [26]. Most likely these variations in the biological efficiency of rat C5a reflect differences in the sensitivity of the cell types or tissues used in the biological assay systems.…”

“…In addition the recombinant protein was not glycosylated. Thus it is possible, that the differences in the biological efficiency of the recombinant rat C5a preparation from the native rat C5a [26] may also partially reflect sequence differences and the lack of glycosylation. However, differences in sequence and glycosylation probably play only a minor role, since recombinant rat C5a and native human C5a were almost equally potent in the release of N-acetyl-fl-D-glucosaminidase from human granulocytes (see below).…”

Induction of Functional Anaphylatoxin C5a Receptors on Hepatocytes by In Vivo Treatment of Rats with IL-6

“…C5a, a glycopeptide of 77 and 74 aa in rat and human, respectively (1)(2)(3)(4), causes the degranulation of mast cells, the contraction of smooth muscle cells, an increase in vascular permeability, and the chemotaxis and activation of neutrophils with local release of reactive oxygen species, eicosanoids, and cytokines (5-7). C5a receptors (C5aR) 2 have been cloned in various species including human (8,9), mouse (10), dog (11), guinea pig (12), and rat (13,14).…”

Anaphylatoxin C5a Actions in Rat Liver: Synergistic Enhancement by C5a of Lipopolysaccharide-Dependent α2-Macroglobulin Gene Expression in Hepatocytes Via IL-6 Release from Kupffer Cells

“…C5a, a glycopeptide of 77 (rat) and 74 (human) amino acids, respectively (1)(2)(3)(4), is the N-terminal cleavage product of the ␣-chain of the precursor protein C5. C5a is involved in systemic defense reactions by causing contraction of smooth muscle cells, an increased vascular permeability, degranulation of mast cells, and activation, chemotaxis, and margination of neutrophils (5)(6)(7).…”