Glycosylation and the complement system.
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Biomedical subjects
B Paul Morgan
Publications and source records attributed to B Paul Morgan.
Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.
BACKGROUND: Decay-accelerating factor (DAF) has inhibitory activity toward complement C3 and C5 convertases. DAF is present in human glomeruli and on cultured human glomerular visceral epithelial cells (GEC). We studied the distribution and function of rat DAF. METHODS: Function-neutralizing antibodies (Abs) were raised against DAF. The distribution of DAF in vivo was determined by immunoelectron microscopy. Functional studies were performed in cultured GEC and following IV injection of anti-DAF Abs into rats. RESULTS: DAF was present exclusively on the apical surfaces of GEC, and was not present on the basal surfaces of GEC, nor other glomerular or kidney cells. DAF was functionally active on cultured GEC, and served to limit complement activation in concert with CD59, an inhibitor of C5b-9 formation. Upon injection into normal rats, anti-DAF F(ab')2 Abs bound to GEC in vivo, yet there was no evidence for complement activation and animals did not develop abnormal albuminuria. Anti-megalin complement-activating IgG Abs were "planted" on GEC, which activated complement as evidenced by the presence of C3d on GEC. Attempts to inhibit DAF function with anti-DAF Abs did not affect the quantity of complement activation by these anti-megalin Abs, nor did it lead to development of abnormal albuminuria. In contrast, in the puromycin aminonucleoside model of GEC injury and proteinuria, anti-DAF Abs slowed the recovery from renal failure that occurs in this model. CONCLUSION: In cultured rat GEC, DAF is an effective complement regulator. In vivo, DAF is present on GEC apical surfaces. Yet, it appears that DAF is not essential to prevent complement activation from occurring under normal circumstances and in those cases in which complement-activating Abs are present on the basal surfaces of GEC in vivo. However, in proteinuric conditions, DAF appears to be protective to GEC.
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Coxsackie B viruses that use human DAF as a receptor infect pig cells via pig CAR and do not use pig DAF.
Coxsackie B viruses (CVB) are enteroviruses belonging to the family Picornaviridae. Serotypes 1, 3 and 5 of CVB bind to the human membrane complement regulator decay-accelerating factor (DAF) and the coxsackievirus/adenovirus receptor (CAR), using either or both as receptors. These viruses are known to infect pig cell lines, but the receptor(s) involved has not been identified. We have recently characterized the pig homologue of DAF and here explore the interactions of human DAF-binding CVB with pig homologues of DAF and CAR. CVB infection of three pig cell lines resulted in cytolysis, which could be not be blocked by anti-pig DAF antibodies. CVB bound to CHO cells transfected with human DAF, but not pig DAF. Modification of pig DAF by incorporation of the fourth short consensus repeat of human DAF did not confer CVB-binding capacity. CVB did bind CHO cells expressing pig or human CAR, and pre-incubation of pig cells with anti-CAR antibody blocked CVB infection.
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Bacterial expression and membrane targeting of the rat complement regulator Crry: a new model anticomplement therapeutic.
Inappropriate or unregulated activation of complement can contribute to pathology in inflammatory diseases. Previous studies have shown that soluble recombinant regulators of complement are effective in animal models and some human diseases. However, limitations include cost, rapid clearance, and unwanted systemic effects. To avoid some of these problems, bacterial expression of regulators has been optimized and methods for the addition of a membrane-targeting moiety to the complement regulator developed. When administered directly to sites of inflammation, membrane-targeted human regulators are retained and inhibit complement-activation locally. To test the efficacy of membrane-targeted complement regulators in vivo, we have undertaken the expression and membrane targeting of the rat-complement regulator Crry. A soluble recombinant form of Crry, containing only the first four short consensus repeats, was expressed in a mammalian expression system and shown to be functional as a fluid phase regulator. To generate the quantities required for testing in vivo, Crry was expressed in bacteria and refolded successfully. Refolded protein had full-complement regulatory activity in vitro. Attachment of a membrane address tag conferred membrane-binding capacity and greatly increased complement regulatory function in vitro. This novel anticomplement agent can now be applied to rat models of arthritis and other inflammatory diseases.
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Roles of the complement system in human neurodegenerative disorders: pro-inflammatory and tissue remodeling activities.
Complement is an important component of the innate immune response with the capacity to recognize and clear infectious challenges that invade the CNS through a damaged blood brain barrier. For instance, the membrane attack complex is involved in cytotoxic and cytolytic activities while other smaller fragments lead to cell activation (chemotaxis) and phagocytosis of the intruders. It is noteworthy that there is a growing body of evidence that uncontrolled complement biosynthesis and activation in the CNS can contribute to exacerbate the neuronal loss in several neurodegenerative disorders. We provide here an insightful review of the double-edged sword activities of the local innate complement system in the CNS and discuss further the potential therapeutic avenues of delivering complement inhibitors to control brain inflammation.
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