ABSTRACT
Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.
Subject(s)
Bacterial Proteins/genetics , Complement Inactivator Proteins/biosynthesis , Genome, Bacterial , Multigene Family , Sesquiterpenes/metabolism , Streptomyces/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Complement Inactivator Proteins/chemistry , Molecular Sequence Data , Molecular Structure , Phylogeny , Sequence Alignment , Sesquiterpenes/chemistry , Streptomyces/chemistry , Streptomyces/classification , Streptomyces/metabolismABSTRACT
BACKGROUND: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. METHODS: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. RESULTS: Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. CONCLUSION: Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.
Subject(s)
Breast Neoplasms/genetics , Complement Inactivator Proteins/genetics , Gene Expression Regulation, Neoplastic , Membrane Proteins/genetics , RNA, Neoplasm/genetics , T-Lymphocytes/metabolism , Adult , Aged , Aged, 80 and over , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Complement Inactivator Proteins/biosynthesis , Female , Humans , Immunohistochemistry , Membrane Proteins/biosynthesis , Microscopy, Fluorescence , Middle Aged , Polymerase Chain Reaction , Prognosis , T-Lymphocytes/pathologyABSTRACT
Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Approximately 50% of AMD patients have a polymorphism in the negative regulator of complement known as Factor H. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroid and retinal pigment epithelium relative to individuals homozygous for the wild-type allele. An inability to form MAC due to a polymorphism in C9 is protective against the formation of choroidal neovascularization (CNV) in AMD patients. Hence, blocking MAC in AMD patients may be protective against CNV. Here we investigate the potential of human proline/arginine-rich end leucine-rich repeat protein (PRELP) as an inhibitor of complement-mediated damage when delivered via the subretinal route using an AAV2/8 vector. In a fluorescence-activated cell sorting (FACS) lysis assay, PRELP inhibited normal human serum-mediated lysis of Hepa-1c1c7 cells by 18.7%. Unexpectedly, PRELP enhanced the formation of tubes by human umbilical vein endothelial cells (HUVECs) by approximately 240%, but, when delivered via an AAV vector to the retina of mice, PRELP inhibited laser-induced CNV by 60%. PRELP reduced deposition of MAC in vivo by 25.5%. Our results have implications for the development of complement inhibitors as a therapy for AMD.
Subject(s)
Choroidal Neovascularization/prevention & control , Complement Inactivator Proteins/genetics , Complement Membrane Attack Complex/antagonists & inhibitors , Extracellular Matrix Proteins/genetics , Glycoproteins/genetics , Macular Degeneration/genetics , Animals , Blindness/genetics , Choroid/blood supply , Choroid/pathology , Choroidal Neovascularization/genetics , Complement Activation , Complement Factor H/genetics , Complement Inactivator Proteins/biosynthesis , Complement Membrane Attack Complex/biosynthesis , Dependovirus/genetics , Disease Models, Animal , Extracellular Matrix Proteins/biosynthesis , Genetic Therapy , Genetic Vectors , Glycoproteins/biosynthesis , HEK293 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Macular Degeneration/prevention & control , Mice , Mice, Inbred C57BL , Polymorphism, Single Nucleotide , Retina/pathology , Retinal Pigment Epithelium/pathologyABSTRACT
Complement and dendritic cells (DCs) are essential components of innate immunity. Both participate in local inflammation and moreover have roles in the initiation of the acquired immunity response and in the maintenance of tolerance. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, Factor I, Factor B and complement receptors 3 and 4. In this study, we demonstrate that human DCs are a source of other soluble complement proteins including C1q, C4b binding protein (C4BP), C7 and C8. Complement receptors (CR)1 and the CD18 chain (common for CR3 and CR4) were also present on DCs while CR2 was not detected.
Subject(s)
Complement Inactivator Proteins/biosynthesis , Complement System Proteins/biosynthesis , Dendritic Cells/metabolism , Monocytes/cytology , Receptors, Complement/biosynthesis , Cell Differentiation/immunology , Cell Lineage/immunology , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , Humans , Immunity, Innate , Immunophenotyping , Macrophages/immunology , Macrophages/metabolism , Monocytes/immunology , Monocytes/metabolismABSTRACT
Activation of each complement initiation pathway (classical, alternative, and lectin) can lead to the generation of bioactive fragments with resulting inflammation in target organs. The objective of the current study was to determine the role of specific complement activation pathways in the pathogenesis of experimental anti-type II collagen mAb-passive transfer arthritis. C57BL/6 mice were used that were genetically deficient in either the alternative pathway protein factor B (Bf(-/-)) or in the classical pathway component C4 (C4(-/-)). Clinical disease activity was markedly decreased in Bf(-/-) compared with wild-type (WT) mice (0.5 +/- 0.22 (n = 6) in Bf(-/-) vs 8.83 +/- 0.41 (n = 6) in WT mice (p < 0.0001)). Disease activity scores were not different between C4(-/-) and WT mice. Analyses of joints showed that C3 deposition, inflammation, pannus, cartilage, and bone damage scores were all significantly less in Bf(-/-) as compared with WT mice. There were significant decreases in mRNA levels of C3, C4, CR2, CR3, C3aR, and C5aR in the knees of Bf(-/-) as compared with C4(-/-) and WT mice with arthritis; mRNA levels for complement regulatory proteins did not differ between the three strains. These results indicate that the alternative pathway is absolutely required for the induction of arthritis following injection of anti-collagen Abs. The mechanisms by which these target organ-specific mAbs bypass the requirements for engagement of the classical pathway remain to be defined but do not appear to involve a lack of alternative pathway regulatory proteins.
Subject(s)
Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Cartilage, Articular/immunology , Cartilage, Articular/pathology , Collagen/immunology , Complement Pathway, Alternative/immunology , Immunization, Passive , Animals , Antibodies, Monoclonal/administration & dosage , Arthritis, Experimental/genetics , Complement C3/chemistry , Complement C4/deficiency , Complement C4/genetics , Complement Factor B/deficiency , Complement Factor B/genetics , Complement Factor H/chemistry , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/genetics , Cytokines/biosynthesis , Cytokines/genetics , Disease Models, Animal , Immunization, Passive/methods , Immunohistochemistry , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/biosynthesis , Synovial Membrane/immunology , Synovial Membrane/metabolism , Synovial Membrane/pathologyABSTRACT
This study was undertaken to explore the role of complement regulatory proteins (CRPs) in experimental autoimmune anterior uveitis (EAAU). We observed that the levels of CRPs, Crry and CD59, in the eyes of Lewis rats increased during EAAU and remained elevated when the disease resolved. The in vivo role of these CRPs in EAAU was explored using neutralizing mAbs, antisense oligodeoxynucleotides (AS-ODNs), and small interfering RNAs against rat Crry and CD59. Suppression of Crry in vivo at days 9, 14, or 19 by neutralizing mAb or AS-ODNs resulted in the early onset of disease, the exacerbation of intraocular inflammation, and delayed resolution. Suppression of CD59 was only effective when the Abs and ODNs were given before the onset of disease. The most profound effect on the disease was observed when a mixture of Crry and CD59 mAbs or AS-ODNs was administered. A similar effect was observed with a combination of Crry and CD59 small interfering RNA. There was no permanent histologic damage to ocular tissue after the inflammation cleared in these animals. Increased complement activation as determined by increased deposition of C3, C3 activation fragments, and membrane attack complex was observed in the eyes of Lewis rats when the function and/or expression of Crry and CD59 was suppressed. Thus, our results suggest that various ocular tissues up-regulate the expression of Crry and CD59 to avoid self-injury during autoimmune uveitis and that these CRPs play an active role in the resolution of EAAU by down-regulating complement activation in vivo.
Subject(s)
Complement Inactivator Proteins/antagonists & inhibitors , Complement Inactivator Proteins/biosynthesis , Down-Regulation/immunology , Uveitis, Anterior/immunology , Uveitis, Anterior/metabolism , Animals , Antibodies, Blocking/administration & dosage , Antibodies, Monoclonal/administration & dosage , Antigens, Surface , Autoantigens/biosynthesis , Autoantigens/immunology , Autoantigens/physiology , CD59 Antigens/biosynthesis , CD59 Antigens/genetics , CD59 Antigens/immunology , CD59 Antigens/physiology , Cattle , Complement C3/metabolism , Complement C3-C5 Convertases/metabolism , Complement Inactivator Proteins/genetics , Complement Inactivator Proteins/physiology , Complement Membrane Attack Complex/metabolism , Down-Regulation/genetics , Male , Melanins/immunology , Melanins/metabolism , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligodeoxyribonucleotides, Antisense/chemical synthesis , RNA, Small Interfering/administration & dosage , Rats , Rats, Inbred Lew , Receptors, Cell Surface , Receptors, Complement/antagonists & inhibitors , Receptors, Complement/biosynthesis , Receptors, Complement/genetics , Receptors, Complement/immunologyABSTRACT
BACKGROUND: The link between the lupus band and pathogenesis remains controversial, because immunoglobulins and complement components, including the membrane attack complex, can be found in both lesional and non-lesional skin of patients with systemic lupus erythematosus (SLE). The expression of proteins that regulate complement has not been previously investigated in the skin of patients with SLE. AIM: The aim of this study is to compare the expression of protectin (CD59), which demonstrates the activation of the classical pathway of complement, in non-lesional skin obtained from patients with SLE with its expression in normal skin. This may help us explain the link between the lupus band and pathogenesis of cutaneous lupus erythematosus. METHODS: An indirect immunofluorescence technique was performed in order to provide unequivocal evidence for the activation of complement via the classical pathway and to compare the expression of CD59 in non-lesional skin from patients with SLE with normal skin samples obtained from healthy people. RESULTS: The activation of the classical pathway of complement was demonstrated in non-lesional skin in more than 90% of SLE patients investigated in this study. Staining intensity of the complement regulatory protein CD59 was markedly increased in the majority of non-lesional skin samples obtained from patients with SLE, compared to that from normals. CONCLUSIONS: CD59 is overexpressed in non-lesional skin in which complement activation has occurred. It seems likely that an increased and continuous CD59 expression may be important for maintaining the integrity of the skin BMZ during inflammatory responses involving complement activation in SLE skin. Alahlafi A, Wordsworth P, Wojnarowska F. Activation/inactivation of the classical pathway of complement in non-lesional skin of patients with systemic lupus erythematosus.
Subject(s)
CD59 Antigens/biosynthesis , Complement Inactivator Proteins/biosynthesis , Lupus Erythematosus, Discoid/metabolism , Lupus Erythematosus, Systemic/metabolism , Skin/metabolism , Fluorescent Antibody Technique, Indirect , Humans , Lupus Erythematosus, Discoid/pathology , Lupus Erythematosus, Systemic/pathology , Skin/pathologyABSTRACT
BACKGROUND: It has previously been reported that there is a strong correlation between the expression of glycosylphosphatidylinositol (GPI)-anchored complement membrane inhibitor in gastric epithelium and the severity of inflammation of gastric mucosa. To investigate the regulation of complement activity in gastric epithelium during Helicobacter pylori (H. pylori)-associated gastritis, the expression of GPI-anchored complement membrane inhibitors, decay-accelerating factor (DAF) and 20-kDa homologous restriction factor 20 (HRF20), and membrane cofactor protein (MCP), which is a transmembrane protein, were evaluated after removal of the H. pylori stimulus. Furthermore, the expression of the complement fragment, C3c, was also investigated. METHODS: Forty-six patients with epigastric symptoms and endoscopically confirmed peptic ulcer or gastritis who had H. pylori infection of the gastric mucosa were enrolled in the present study. Biopsy specimens were obtained from the gastric antrum and corpus 1 month before and after eradication. Helicobacter pylori infection was determined by the rapid urease test, histology, and culture before eradication, and by histology, culture, and urea breath test after eradication. Gastric biopsy specimens obtained before and after eradication were evaluated for infiltration by neutrophils and mononuclear cells. The expression of complement membrane inhibitors, DAF, HRF20, and MCP and that of the main complement fragment, C3c, was immunohistochemically evaluated. RESULTS: One month after the eradication of H. pylori, the infiltration by neutrophils and mononuclear cells in the gastric mucosa decreased significantly (P < 0.0001) as compared with that before eradication. The expression of DAF, HRF20, and C3c on gastric mucosal epithelium also significantly decreased in both the antrum and the corpus (P < 0.05) 1 month after eradication. However, no change was observed in the expression of MCP. CONCLUSIONS: The decrease in the expression of GPI-anchored complement regulator and the complement after removal of a chronic microbial stimulus suggests that the gastric epithelium appears to undergo an aggressive stress of complement during H. pylori infection. Conclusively, DAF and HRF20 may play an important protective role against complement-mediated damage induced by chronic microbial stimuli in such a pathological condition.
Subject(s)
Complement System Proteins/immunology , Gastric Mucosa/immunology , Helicobacter Infections/immunology , Helicobacter pylori , Amoxicillin/therapeutic use , Anti-Bacterial Agents/therapeutic use , Anti-Ulcer Agents/therapeutic use , CD55 Antigens/biosynthesis , CD59 Antigens/biosynthesis , Clarithromycin/therapeutic use , Complement C3c/immunology , Complement Inactivator Proteins/biosynthesis , Female , Gastric Mucosa/physiopathology , Gastritis/drug therapy , Gastritis/immunology , Glycosylphosphatidylinositols/biosynthesis , Helicobacter Infections/drug therapy , Humans , Male , Membrane Cofactor Protein/biosynthesis , Middle Aged , Omeprazole/therapeutic use , Peptic Ulcer/drug therapy , Peptic Ulcer/immunology , Receptors, Complement/biosynthesisABSTRACT
Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.
Subject(s)
Blood Proteins/physiology , C-Reactive Protein/metabolism , Complement C3-C5 Convertases/antagonists & inhibitors , Complement Factor H/physiology , Complement Inactivator Proteins/physiology , Heparin/metabolism , Lipoproteins, HDL/metabolism , Blood Proteins/biosynthesis , Blood Proteins/genetics , Blood Proteins/metabolism , Complement C3-C5 Convertases/metabolism , Complement C3b/metabolism , Complement Factor H/metabolism , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/genetics , Complement Inactivator Proteins/metabolism , Complement System Proteins , Consensus Sequence , Fibrinogen/physiology , Humans , Hydrolysis , Lipoproteins, HDL/blood , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Pichia/genetics , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Repetitive Sequences, Amino AcidABSTRACT
We have previously described the activation of the classical, alternative, and terminal complement cascade pathways after acute contusion spinal cord injury using the New York University (NYU) weight-drop impactor. In the present study, we examined the induction of protein regulators of the complement cascade, factor H (FH), and clusterin, in the same experimental paradigm. The spinal cord of laminectomized adult rats was subjected to mild or severe injury using impactor weight-drop heights of 12.5 and 50 mm, respectively. The spinal cords of control and injured animals were evaluated at 1, 7, and 42 days after injury. Immunocytochemistry revealed a robust increase in the numbers and intensity of staining of FH, and clusterin-positive cells in the injured cord at all three time points, with the highest increases observed at 1 and 42 days after injury. FH and clusterin-positive cells were observed among neurons as well as oligodendrocytes. The increased expression was detected both rostrally and caudally from the injury site, in the latter case at distances up to 20 mm. The precise biological significance of injury-induced upregulation of these proteins remains to be determined. However, FH and clusterin are potent regulators of complement activity targeting upstream (FH) and downstream (clusterin) molecules of the pro-inflammatory cascade, which could be of vital importance in preventing a "runaway" inflammatory reaction in the injured spinal cord.
Subject(s)
Complement Inactivator Proteins/biosynthesis , Neuroglia/metabolism , Neurons/metabolism , Spinal Cord Injuries/metabolism , Up-Regulation/physiology , Animals , Neuroglia/pathology , Neurons/pathology , Rats , Rats, Long-Evans , Spinal Cord Injuries/pathology , Thoracic VertebraeABSTRACT
Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.
Subject(s)
Complement C5a/antagonists & inhibitors , Complement C5a/metabolism , Complement Inactivator Proteins/chemistry , Complement Pathway, Alternative/immunology , Complement Pathway, Classical/immunology , Ornithodoros/immunology , Salivary Proteins and Peptides/chemistry , Amino Acid Sequence , Animals , Cloning, Molecular , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/genetics , Humans , Inhibitory Concentration 50 , Molecular Sequence Data , Ornithodoros/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Salivary Proteins and Peptides/biosynthesis , Salivary Proteins and Peptides/genetics , Salivary Proteins and Peptides/isolation & purification , Sequence AlignmentABSTRACT
NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.
Subject(s)
CD55 Antigens/physiology , Complement Inactivator Proteins/physiology , Cytotoxicity, Immunologic , Down-Regulation/immunology , Killer Cells, Natural/immunology , Amino Acid Substitution/genetics , Animals , Blotting, Western , CD55 Antigens/biosynthesis , CD55 Antigens/blood , CD55 Antigens/genetics , Cell Line , Chromium Radioisotopes/metabolism , Complement Activation/genetics , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/genetics , Consensus Sequence/genetics , Cytotoxicity Tests, Immunologic/methods , Cytotoxicity, Immunologic/genetics , Down-Regulation/genetics , Fibroblasts/immunology , Fibroblasts/metabolism , Flow Cytometry , Humans , Interferon-gamma/metabolism , K562 Cells , Killer Cells, Natural/metabolism , Mutagenesis, Site-Directed , Repetitive Sequences, Amino Acid/genetics , Sequence Deletion , Swine , TransfectionABSTRACT
Exo-polymer (EP) was produced at 1.2 g l(-1) in submerged culture of Auricular auricula-judae. Crude EP (AJ-0) has 70% anti-complementary activity (inhibition of total complementary hemolysis 50%; ITCH50). The activating pathway of the complement system occurred through both the classical and alternative pathways, though the major pathway was the classical one. Fractionation of AJ-0 using Sepharose CL-6B gel chromatography gave three major fractions (AJ-Fr-I, II and III) of which the first was the most active. The mycelial growth and EP production of A. auricula-judae were optimal at pH 6, 25 degrees C and pH 5, 25 degrees C, respectively.
Subject(s)
Biopolymers/biosynthesis , Biopolymers/immunology , Cell Culture Techniques/methods , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/immunology , Spiroplasma/growth & development , Spiroplasma/metabolism , Biopolymers/chemistry , Complement Inactivator Proteins/chemistryABSTRACT
Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.
Subject(s)
Complement C3/biosynthesis , Complement C3/chemistry , Complement C5/biosynthesis , Complement C5/chemistry , Receptors, Cell Surface/biosynthesis , Recombinant Proteins/chemistry , Amino Acid Motifs/genetics , Amino Acid Sequence , Binding, Competitive/immunology , Complement C3/genetics , Complement C5/genetics , Complement C5/metabolism , Complement C6/metabolism , Complement C7/metabolism , Complement Hemolytic Activity Assay/methods , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/chemistry , Cysteine/chemistry , Disulfides/chemistry , Escherichia coli/genetics , Humans , Molecular Sequence Data , Netrin Receptors , Plasmids , Protein Binding/immunology , Protein Structure, Secondary , Protein Structure, Tertiary/genetics , Protein Subunits/biosynthesis , Protein Subunits/chemistry , Protein Subunits/genetics , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolismABSTRACT
In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.
Subject(s)
Antigens, CD/biosynthesis , CD55 Antigens/biosynthesis , CD59 Antigens/biosynthesis , Complement Inactivator Proteins/biosynthesis , Endometrium/immunology , Membrane Glycoproteins/biosynthesis , Antigens, CD/immunology , Autoantibodies/blood , Autoantibodies/immunology , Blotting, Western , CD55 Antigens/immunology , CD59 Antigens/immunology , Complement Inactivator Proteins/immunology , Electrophoresis, Polyacrylamide Gel , Endometrium/pathology , Female , Flow Cytometry , Gene Expression Regulation/immunology , Humans , Infertility, Female/immunology , Lipopolysaccharides/immunology , Membrane Cofactor Protein , Membrane Glycoproteins/immunology , Membrane Proteins/immunology , Microscopy, Fluorescence , Tumor Cells, CulturedABSTRACT
Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.
Subject(s)
Bacterial Proteins/biosynthesis , Blood Bactericidal Activity/immunology , Borrelia burgdorferi/growth & development , Borrelia burgdorferi/immunology , CD59 Antigens/biosynthesis , Complement Inactivator Proteins/biosynthesis , Complement System Proteins/toxicity , Cytotoxicity, Immunologic , Antibodies, Blocking/metabolism , Antibodies, Blocking/pharmacology , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Binding Sites, Antibody , Borrelia burgdorferi/metabolism , Borrelia burgdorferi/ultrastructure , CD59 Antigens/immunology , CD59 Antigens/metabolism , CD59 Antigens/ultrastructure , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Complement C3/metabolism , Complement C7/metabolism , Complement C8/metabolism , Complement C9/metabolism , Complement Inactivator Proteins/immunology , Complement Inactivator Proteins/metabolism , Complement Inactivator Proteins/ultrastructure , Complement Membrane Attack Complex/antagonists & inhibitors , Fluorescent Antibody Technique, Direct , Humans , Immunity, Innate , Species Specificity , Trypsin/pharmacologyABSTRACT
A secreted, hypervariable virulence factor called the streptococcal inhibitor of complement (Sic) has been linked to the reemergence of epidemics due to the human pathogenic bacterium Group A Streptococcus. This paper describes a method for expressing and purifying Sic from an attenuated GAS strain using a chemically defined growth medium. This method was used to label specific amino acid residue types in Sic with forms containing the magnetically active isotope (15)N, at the amide nitrogen. The (15)N-labeling of Sic permits a detailed investigation of the structure and dynamics of the protein using nuclear magnetic resonance spectroscopy. The level of stable isotope incorporation was established using mass spectrometry and nuclear magnetic resonance spectroscopy.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/chemistry , Culture Media/chemistry , Streptococcus pyogenes/metabolism , Amides/chemistry , Bacterial Proteins/isolation & purification , Complement Inactivator Proteins/isolation & purification , Humans , Isotope Labeling/methods , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular/methods , Spectrometry, Mass, Electrospray Ionization , Streptococcus pyogenes/genetics , Streptococcus pyogenes/pathogenicity , VirulenceABSTRACT
To investigate the role of complement in lupus nephritis, we used MRL/lpr mice and a transgene overexpressing a soluble complement regulator, soluble CR1-related gene/protein y (sCrry), both systemically and in kidney. Production of sCrry in sera led to significant complement inhibition in Crry-transgenic mice relative to littermate transgene negative controls. This complement inhibition with sCrry conferred a survival advantage to MRL/lpr mice. In a total of 154 animals, 42.5% transgene-negative animals had impaired renal function (blood urea nitrogen > 50 mg/dl) compared with 16.4% mice with the sCrry-producing transgene (p < 0.001). In those animals that died spontaneously, MRL/lpr mice with the sCrry-producing transgene did not die of renal failure, while those without the transgene did (blood urea nitrogen values of 46.6 +/- 9 and 122 +/- 29 mg/dl in transgene-positive and transgene-negative animals, respectively; p < 0.001). Albuminuria was reduced in those transgenic animals in which sCrry expression was maximally stimulated (urinary albumin/creatinine = 12.4 +/- 4.3 and 36.9 +/- 7.7 in transgene-positive and transgene-negative animals, respectively; p < 0.001). As expected in the setting of chronic complement inhibition, there was less C3 deposition in glomeruli of sCrry-producing transgenic mice compared with transgene-negative animals. In contrast, there was no effect on glomerular IgG deposition, levels of anti-dsDNA Ab and rheumatoid factor, or spleen weights between the two groups. Thus, long-term complement inhibition reduces renal disease in MRL/lpr mice, which translates into improved survival. MRL/lpr mice in which complement is inhibited still have spontaneous mortality, yet this is not from renal disease.
Subject(s)
Complement Inactivator Proteins/biosynthesis , Complement Inactivator Proteins/genetics , Lupus Nephritis/immunology , Lupus Nephritis/mortality , Receptors, Complement/biosynthesis , Receptors, Complement/genetics , Transgenes/immunology , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Complement C3/metabolism , Complement Inactivator Proteins/physiology , Immunoglobulin G/metabolism , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Lupus Nephritis/pathology , Lupus Nephritis/prevention & control , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Transgenic , Receptors, Complement/physiology , Receptors, Complement 3b , Survival AnalysisABSTRACT
Membrane-bound complement inhibitors protect host cells from inadvertent complement attack, and complement inhibitors are often up-regulated on tumors, possibly representing a selective adaptation by tumors to escape elimination by a host antitumor immune response. Relevant in vivo studies using rodent models of human cancer have been hampered by the fact that human complement inhibitors are not effective against rodent complement. Using nude rats and MCF7 cells expressing different rat complement inhibitors, a model of human breast cancer was established to investigate the role of complement and complement inhibitors in tumor progression. Expression of rat CD59, an inhibitor of the terminal cytolytic membrane attack complex of complement, had no effect on the incidence or growth rate of MCF7 tumors. In contrast, expression of rat Crry, an inhibitor of complement activation, dramatically enhanced the tumorigenicity of MCF7 cells. The expression of rat Crry on MCF7 inhibited the in vivo deposition of complement C3 fragments that serve as opsonins for receptors on phagocytes and natural killer cells. These data provide direct in vivo evidence that an inhibitor of complement activation can facilitate tumor growth by modulating C3 deposition. These data indicate an important role for complement opsonization in promoting cell-mediated antitumor immune function, a conclusion further supported by the demonstration that expression of rat Crry, but not rat CD59, on MCF7 cells inhibits rat cell-mediated cytotoxicity in vitro. Rat complement activation on MCF7 tumors was mediated by tumor-reactive antibodies present in the serum of naïve nude rats, but there was also an IgM response to MCF7 tumors, a situation with similarities to some human cancers. These data support a hypothesis that blocking complement inhibitor function on tumor cells will not only enhance monoclonal antibody-mediated immunotherapy but may also be effective at enhancing a normally ineffective humoral immune response in the absence of administered antitumor antibody.
Subject(s)
Breast Neoplasms/pathology , Complement Inactivator Proteins/physiology , Complement System Proteins/metabolism , Mammary Neoplasms, Experimental/pathology , Animals , Antigens, Surface , Breast Neoplasms/immunology , Breast Neoplasms/metabolism , CD59 Antigens/biosynthesis , CD59 Antigens/genetics , CD59 Antigens/physiology , Cell Division/physiology , Complement Activation/physiology , Complement Inactivator Proteins/biosynthesis , Complement System Proteins/physiology , Disease Models, Animal , Female , Humans , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/metabolism , Rats , Receptors, Cell Surface , Receptors, Complement/biosynthesis , Receptors, Complement/genetics , Receptors, Complement/physiology , TransfectionABSTRACT
INTRODUCTION AND OBJECTIVES: Progression of prostate cancer to androgen independence (AI) results in part from the upregulation of anti-apoptotic genes following androgen withdrawal, and androgen-independent disease remains the primary obstacle to improved survival. Testosterone-repressed prostate message-2 (TRPM-2) encodes the anti-apoptotic protein clusterin, which is upregulated in response to cellular compromise as observed in normal and malignant tissues undergoing apoptosis. Systemic administration of antisense clusterin oligonucleotides in prostate cancer xenograft models delays progression to AI and enhances chemosensitivity. The objective of this study was to define changes in clusterin expression following neoadjuvant hormone therapy (NHT) in prostate cancer patients. MATERIALS AND METHODS: Archival radical prostatectomy (RP) specimens were obtained for 128 patients who received either no NHT or treatment for 2-8 weeks, 3 months, or 8 months. Paired needle biopsy specimens were acquired for 30 patients and all tissues were subjected to clusterin immunohistochemistry. Western blot analysis was performed on frozen tissue from 5 untreated and 5 treated patients. RESULTS: Clusterin expression in malignant prostatic tissue was significantly greater in patients who underwent preoperative NHT (P < 0.001). Needle biopsies obtained prior to NHT consistently demonstrated lower staining intensity than corresponding RP specimens (P < 0.001). Western blot analysis confirmed clusterin levels increased 17-fold beginning within 4 weeks after androgen withdrawal. CONCLUSIONS: Upregulation of clusterin levels following androgen ablation therapy may represent an adaptive cell survival response following apoptotic signals like androgen withdrawal. These findings support clusterin as a valid therapeutic target in strategies employing novel multimodality therapy for advanced prostate cancer.