Nuclear magnetic resonance spectroscopy shows the structure of human anaphylatoxin C5a to be a four-helix bundle; the four α-helical segments juxtapose in an antiparallel topology, which are stabilized by three disulfide bonds (Cys21-Cys47, Cys22-Cys54 and Cys34-Cys55) and connected by three peptide loops. The flexible carboxyl terminal tail (aa 69-74 MQLGR) forms a helical turn that is connected by a short loop, which is important for effector functions of C5a. When this region is missing, C5a still binds to the receptor but loses agonistic activity. This and other data support a 'two-site binding' model for C5a-C5aR interaction. The model suggests that the N terminus and disulphide-linked core region of C5a interact with a ‘recognition site’ that includes both the N terminus and the third extracellular loop of C5aR, and the C-terminal region of C5a fits into a binding pocket around the fifth transmembrane region, called the 'effector site'. This model explains why C5a without the C-terminal region loses full effector function, even though it binds to the receptor.
C5a is one of the most potent inflammatory peptides and shows diverse activities on many cell types. Depending on the cell type, C5a signaling can lead to various outcomes including phagocytosis, degranulation, H2O2 production, granule enzyme release, delay or enhancement of apoptosis, chemokine and cytokine production, and chemotaxis.
The potent inflammatory functions of C5a indicate that inhibition of this ligand, or its receptor(s), might alleviate certain inflammatory conditions. There are two receptors known to bind to C5a; C5aR (CD88) and C5L2 (GPR77). Both are seventh transmembrane proteins and their genes are located on chromosome 19, q13.33-13.34 (human) directly neighboring each other. They cluster together with the genes for other closely related chemoattractant receptors such as formyl-peptide receptors FPRH1 and FPRH2. C5aR was the first anaphylatoxin receptor to be cloned, in 1991. In contrast, C5L2 was cloned much later, in 2000, by PCR amplification using degenerate primers based on amino-acid sequences known to be conserved in chemoattractant receptors.The C5L2 sequence shows closest identity to that of C5aR (58%) and C3aR.
C5a and C5a desArg have been reported as ligands for C5L2. Okinaga et al. performed competitive ligand-binding assays with 125I-C5a in cell lines transfected with human C5aR or C5L2. They showed that C5a bound to both C5aR and C5L2 with high affinity (Kd 3.4 and 2.5 nm, respectively). C5a desArg bound to C5aR with greatly reduced affinity (660 nm) compared with C5a, but it bound to C5L2 with affinity as high as C5a (12 nm). Whether C3a and C3a desArg serve as ligands for C5L2 is uncertain, as different laboratories have produced conflicting data.
C5aR was initially thought to be expressed mainly on leukocytes such as neutrophils, eosinophils, basophils, monocytes, dendritic cells and mast cells, but it is now established that C5a receptor can be widely expressed, on both immune and nonimmune cells. Reported nonimmune cell that express C5a receptor include vascular endothelial cells, cardiomyocytes, astrocytes, microglia, neural stem cells, oligodendrocytes, synoviocytes, articular chondrocytes, renal glomerular mesangial cells, hepatic kupfer cells and stimulated hepatocytes, bronchial epithelial cells and keratinocytes.
C5L2 is expressed at much lower levels on both immune and nonimmune cells, compared with C5aR. C5L2 is expressed on neutrophils, macrophages,immature dendritic cells and some nonimmune type cells such as adipocytes and skin fibroblasts,as well as adrenal gland, spinal cord, thyroid, liver, lung, spleen, brain and heart.
Both C5aR and C5L2 are seven transmembrane proteins that belong to rhodopsin-like family. Although these two receptors share structural similarity, there are several important differences. Anaphylatoxin C5a receptor signaling pathways have been studied extensively. C5a receptor couples to pertussis toxin-sensitive Giα2, Giα3 or pertussis toxin-insensitive Gα16 and initiates several downstream signaling pathways. Signaling of C5aR involves intracellular calcium mobilization, and activation of different pathways such as phosphatidylinositol-bisphosphate-3-kinase/Akt (also known as protein kinase-B; PKB),Ras/B-Raf/mitogen-activated protein kinase/extracellular signal-related kinase,phospholipase D (PLD),protein kinase C,p21-activated kinases, which are downstream effectors of cdc42 and rac GTPases,signal transducers and activators of transcription, sphingosine kinase and NF-κB.
As anaphylatoxin C5a is such a potent pro-inflammatory mediator, it is very important to control expression to allow rapid responses to pathogens, but at the same time protection for the host against unregulated overactivity. When this tightly regulated balance is disrupted, overproduction of C5a can occur, leading to uncontrolled inflammation. Excessive production of C5a can downregulate immune responses in some leukocytes, and, at the same time, it can overactivate other cell types.
High levels of C5a potentiate macrophage responses to lipopolysaccharide, to produce excessive levels of pro-inflammatory mediators such as TNF-α and various other chemokines leading to uncontrolled inflammation. On endothelial cells, high levels of C5a leads to enhanced production of pro-inflammatory mediators such as IL-8, IL-1β and RANTES, and tissue factor, which serves as a cofactor for blood coagulation.
Overproduced C5a or upregulated C5a Receptor expression has been implicated in the pathogenesis of many inflammatory conditions, autoimmune and neurodegenerative diseases. These include rheumatoid arthritis, respiratory distress syndrome, inflammatory bowel diseases, glomerulonephritis, systemic lupus erythematosus, ischemia/reperfusion injury, chronic obstructive pulmonary disease, sepsis, multiple sclerosis, asthma and allergy, atherosclerosis, xenograft rejection, hemorhagic shock and antiphospholipid syndrome.
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