Description
General Description
Natural human C3a is prepared by cleavage of human C3 protein by a human C3 convertase. C3a is a member of the anaphylatoxin family of three proteins (C3a, C4a and C5a) produced by the activation of complement. It is an glycosylated polypeptide containing 77 amino acids with a molecular mass of 9,089 Daltons. Many of the biological functions of C3a are similar to those of C5a, but C3a is approximately 10- to 20-fold less active per microgram than C5a. C3a mediates many inflammatory responses including smooth muscle contraction, vasodilation, increased vascular permeability, and release of histamine from mast cells and basophils (Law, S.K.A. and Reid, K.B.M. (1995)). In contrast to C5a, C3a does not exhibit significant neutrophil stimulating activities and does not induce chemotaxis, granule release or superoxide production. C3a acts through the C3a Receptor (C3aR) which is a G-protein coupled receptor found widely distributed on peripheral tissues, lymphoid cells (neutrophils, monocles, and eosinophils) and in the central nervous system (astrocytes, neurons and glial cells) (Law, S.K.A. and Reid, K.B.M. (1995)). These activities of C3a are inactivated by removal of the C-terminal arginine and this occurs rapidly in plasma due to the action of carboxypeptidase N (Meuller-Ortiz, S.L., et al. (2009)).
Physical Characteristics & Structure
Molecular weight: 9,089 calculated molecular mass. Observed mass (MALDI-TOF) is 9092 + 9 mass units. Amino acid sequence (77 amino acids): SVQLTEKRMD KVGKYPKELR KCCEDGMREN PMRFSCQRRT RFISLGEACK KVFLDCCNYI TELRRQHARA SHLGLAR
X-ray-derived crystal structure: Huber, R. et al. (1980) NMRderived structure: Nettesheim, D.G. et al. (1988); Murray, I. et al. (1999).
Function
See General Description above. Although C3a has some measurable chemotactic activity it is hundreds of times less active than C5a in this regard. Due to these differences the role of C3a in this response is thought to be negligible in vivo. However, considering that hundreds of times more C3a than C5a is usually produced during complement activation, especially by alternative pathway activation, this belief may not be entirely correct (see sections below titled In vivo and Regulation). Both C3a and C5a, which are generally released together, can cause anaphylactic shock which is a generalized circulatory collapse similar to that caused by a strong allergic reaction.
Assays
Two well established assays for C3a (and C5a) functional activities include induction of contraction in the guinea pig ileum and the permeation of a dye such as trypan blue from the vasculature into skin. The anaphylatoxins also induce mast cell degranulation, (measured as histamine release), platelet aggregation, IL-1 release from monocytes and the release of prostaglandins and leukotrienes from many cells and tissues. More practical assays include ATP release from guinea pig platelets, serotonin release from guinea pig platelets, N-acetyl-beta-D-glucosidase release from differentiated U937 cells and calcium release from differentiated U937 cells. These assays have been described in detail (Dodds, A.W. and Sim, R.B. (1997)). ELISA kits for the assay of C3a levels (or more correctly C3a desArg levels) in blood and other fluids are sold by many companies. A radioimmunoassay for C3a/C3a desArg is also available. These measurements are useful for detecting complement activation in vivo, but the interpretation of their meaning is complicated by the fact that clearance of the anaphylatoxins is rapid.
In vivo
Freshly drawn normal human serum contains approximately 17 nM C3a (corresponding to activation of about 0.3 % of the total C3). Although this may represent the resting concentration in vivo it is difficult to draw or store blood without some C3 activation so a true in vivo concentration is difficult to determine. The presence of EDTA and Futian in the collection tubes can minimize this background (Pfeifer, P.H. et al. (1999)). Full activation of all C3 in blood (1200 µg/mL) would result in ~6,600 nM C3a (~60 µg/mL). Due to the sensitivity of many C3a responses and the fact that there is 20-fold more C3 than C5 in blood, a biological response can theoretically be initiated by activation of approximately 1/10,000 of the C3 in a local area.
Regulation
C3a levels are regulated by three processes: formation, inactivation and clearance. The enzymes that cleave C3 and release C3a (collectively called C3/C5 convertases) do so at a rate approximately 300-times the rate that these enzymes cleave C5 (Pang burn, M.K. and Müller-Eberhard, H.J. (1986); Rawal, N. and Pang burn, M.K. (2001)). Thus, although C3a is 10 to 20 times less active than C5a for most of the biological activities they share (see General Description above), C3a is produced at a much faster rate. C3a is “inactivated” by removal of its C-terminal arginine amino acid. The product C3a desArg (or C3a without the C-terminal arginine) is produced by the action of the plasma enzyme carboxypeptidase N (Mueller-Ortiz S.L. et al. (2009)). The inactivation is rapid and most C3a is converted to C3a desArg within minutes of its formation. “Inactivated” C3a still possesses some biological activities, but it is considered inactive for most C3a-specific functions. C3a desArg does, however, possess numerous activities of its own including its identification as the acylation-stimulating protein (ASP) released by adipose tissue. This function attributed to C3a desArg controls lipid metabolism through the receptor C5L2 (see C3a desArg product description). Because of the large number of cells bearing C3a receptors (endothelial, immune, smooth muscle, neuronal, etc.) the capture, internalization and digestion of C3a and C3a desArg results in its removal from circulation.