Proteomics of Urinary Vesicles Links Plakins and Complement to Polycystic Kidney Disease.

Novel therapies in autosomal dominant polycystic kidney disease (ADPKD) signal the need for markers of disease progression or response to therapy. This study aimed to identify disease-associated proteins in urinary extracellular vesicles (uEVs), which include exosomes, in patients with ADPKD. We performed quantitative proteomics on uEVs from healthy controls and patients with ADPKD using a labeled approach and then used a label-free approach with uEVs of different subjects (healthy controls versus patients with ADPKD versus patients with non-ADPKD CKD). In both experiments, 30 proteins were consistently more abundant (by two-fold or greater) in ADPKD-uEVs than in healthy- and CKD-uEVs. Of these proteins, we selected periplakin, envoplakin, villin-1, and complement C3 and C9 for confirmation because they were also significantly overrepresented in pathway analysis and were previously implicated in ADPKD pathogenesis. Immunoblotting confirmed higher abundances of the selected proteins in uEVs from three independent groups of patients with ADPKD. Whereas uEVs of young patients with ADPKD and preserved kidney function already had higher levels of complement, only uEVs of patients with advanced stages of ADPKD had increased levels of villin-1, periplakin, and envoplakin. Furthermore, all five proteins correlated positively with total kidney volume. Analysis in kidney tissue from mice with kidney-specific, tamoxifen-inducible Pkd1 deletion demonstrated higher expression in more severe stages of the disease and correlation with kidney weight for each protein of interest. In summary, proteomic analysis of uEVs identified plakins and complement as disease-associated proteins in ADPKD. These proteins are new candidates for evaluation as biomarkers or targets for therapy in ADPKD.

Soluble complement receptor type 1 inhibits complement system activation and improves motor function in acute spinal cord injury.

STUDY DESIGN: Spinal cord injured rat model, treated with soluble complement receptor type 1 (sCR1). SETTING: Experimental Animal Department of China Medical University, Shenyang, China. OBJECTIVES: Soluble CR1 is a powerful inhibitor of complement activation. In this study, we investigate the effectiveness of sCR1 on spinal cord injury (SCI) in rats. METHODS: Spinal cord injury was induced in Sprague-Dawley rats. Three experimental groups were examined; the sCR1 group was administered sCR1 at 1 h after the SCI, whereas the control group was administered saline at 1 h after SCI and the sham group underwent a sham operation without SCI or administration. The expressions of C9 and CD59 in the injured spinal cords were evaluated by immunohistochemistry, and numbers of positive cells counted. Furthermore, myeloperoxidase (MPO) activity and motor function were evaluated in each group. RESULTS: At all postoperative time points, the numbers of C9- and CD59-positive cells in the sCR1 group were reduced compared with the control group and MPO activity was significantly decreased compared with both other groups. Moreover, the Basso, Beattie and Bresnahan score for the sCR1 group was significantly improved as compared with that of the control group after 7 days postoperatively. CONCLUSION: Soluble CR1 decreases inflammation reactions by inhibiting activation of the complement system and improves motor function after acute SCI.

Molecular aspects of complement-mediated bacterial killing. Periplasmic conversion of C9 from a protoxin to a toxin.

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.

Function of the factor I modules (FIMS) of human complement component C6.

In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex.

Complement resistance in Borrelia burgdorferi strain 297: outer membrane proteins prevent MAC formation at lysis susceptible sites.

Two variants of Borrelia burgdorferi strain 297, complement-resistant wild-type (WT297) and complement-sensitive mutant (MUT297), were used as a model to study the mechanism of resistance to the alternative complement pathway in this organism. No difference in the quantity of membrane attack complex (MAC) deposition on WT297 and MUT297 was observed after 2 h incubation with normal human serum (NHS), at which time 4% of WT297 and 95% of MUT297 were killed. The polymerization of C9 bound to WT297 and MUT297 was demonstrated by immunoblotting using an anti-C9 polyclonal antibody. Immunofluorescence and thin-section immunoelectron microscopy showed MAC to be diffusely distributed on the outer membrane of both variants. Furthermore, MAC appeared to be tightly bound to the surface of both variants as demonstrated by elution studies. Protease treatment rendered WT297 susceptible to killing by NHS, suggesting that outer membrane proteins may be associated with complement resistance of WT297. One- and two-dimensional gel electrophoreses showed that proteins of 20 and 30 kDa, and 66 kDa were present in WT297 but were absent or sparse in trypsin-treated WT297 and MUT297. Interestingly, immunoblotting using a polyclonal antibody against C3 showed that C3 fragments appeared to bind different acceptors on WT297 than on trypsin-treated WT297, or MUT297. Therefore, the binding of C3 fragments to acceptors on WT297, in contrast to MUT297, may not direct the formation of the MAC to lysis-susceptible sites on the surface of the bacterium, resulting in the complement resistance of WT297.

Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization.

Normal endochondral bone formation follows a temporal sequence: immature or resting chondrocytes move away from the resting zone, proliferate, flatten, become arranged into columns, and finally become hypertrophic, disintegrate, and are replaced by bone. The mechanisms that guide this process are incompletely understood, but they include programmed cell death, a stage important in development and some disease processes. Using immunofluorescence we have studied the distribution of various complement proteins to examine the hypothesis that this sequence of events, particularly cell disintegration and matrix dissolution, are complement mediated. The results of these studies show that complement proteins C3 and Factor B are distributed uniformly in the resting and proliferating zones. Properdin is localized in the resting and hypertrophic zone but not in the proliferating zone. Complement proteins C5 and C9 are localized exclusively in the hypertrophic zones. This anatomically segregated pattern of distribution suggests that complement proteins may be important in cartilage-bone transformation and that the alternate pathway is involved.

Isolation of the C9b fragment of human complement component C9 using urea in the absence of detergents.

The bactericidal activity of the C5b-9 complex of complement is dependent upon the terminal complement component C9. The precursor C5b-8 complex is not harmful to bacterial cells until C9 is added to complete the C5b-9 complex. The C9 molecule can be proteolytically cleaved by thrombin to yield an intact, nicked molecule that remains fully functional when added to either bacterial cells or erythrocytes bearing pre-formed C5b-8 complexes. In investigating the membranolytic function of C9 in the C5b-9 complex, the carboxyl-terminal portion of the nicked molecule (C9b) has been shown to be membranolytic when added to erythrocytes, liposomes, or bacterial inner membranes in the absence of any other complement components. The isolation of C9b from nicked C9 has been accomplished by preparative gel electrophoresis using detergents, however the study of the activity of C9b in membrane systems may be complicated by the possible presence of residual detergent. To address this concern, we have used 4 M urea in conjunction with hydroxyapatite chromatography and a phosphate elution procedure to separate the domains of nicked C9. The isolated C9b domain, free of detergents and in the absence of any other complement components, was found to be membranolytic. C9b isolated in this manner was capable of lysing erythrocytes and inhibiting the growth of bacterial spheroplasts.

The functions of the ninth component of human complement are sustained by disulfide bonds with different susceptibilities to reduction.

Purified C9 with expected hemolytic and polymerizing activities was found to contain approximately 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intra-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on the functions of C9. On the other hand, reduction of C9 by 1 mM dithiothreitol (DTT) (6-fold molar excess over Cys residues) followed by alkylation resulted in a complete block of polymerization activity and a 50% loss of C9 hemolytic activity. In contrast, the ability of C9 to bind EAC1-8 remained largely unaffected. The loss of poly-C9 formation activity correlated with the alkylation of approx. 6 liberated sulfhydryl groups. Hemolytic activity was abolished by treatment with > 5 mM DTT which allowed the liberation of approximately 18 sulfhydryl groups. Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functions of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with different susceptibility to reducing reagents. The maintenance of the N-terminal C9a region is essential for polymerization, but not EAC1-8 binding activity of C9. Taken together, the results of the present study differentiate in molecular terms several of the functional portions of C9, and stress the significance of intra-chain disulfide linkages in maintaining the structural components necessary for the functions of C9.

Supplemental complement component C9 enhances the capacity of neonatal serum to kill multiple isolates of pathogenic Escherichia coli.

Previous studies demonstrated that, compared with adult serum, neonatal serum contained a diminished concentration of complement component C9 and that supplemental C9 enhanced the capacity of neonatal serum to kill an isolate of Escherichia coli. Therefore, experiments were designed to determine the mechanisms by which supplemental C9 enhances the bactericidal capacity of neonatal serum and to determine whether supplemental C9 enhances the capacity of neonatal serum to kill several different pathogenic strains of E. coli. A radiobinding assay and immunogold electron microscopy using a monoclonal anti-C9 antibody revealed that, compared with 40% adult serum, neonatal serum deposited a diminished quantity of C9 onto E. coli O7w:K1:NM. Supplemental C9 (75 mg/L) significantly enhanced the quantity of C9 deposited by the neonatal serum. Treatment with 10 mM MgEGTA (a mixture of 100 mM MgCl2 and 100 mM EGTA that blocks activation of the classic complement pathway but leaves the alternative pathway intact) abolished the capacity of neonatal serum to deposit C9 and to kill the bacteria. Supplemental C9 enhanced the capacity of neonatal serum to kill eight different blood isolates of E. coli. Therefore, supplemental C9 enhanced the capacity of neonatal serum to kill E. coli by increasing the total quantity of C9 deposited via activation of the classic complement pathway. Neonatal serum contained sufficient quantities of classic pathway components, other than C9, to deposit the supplemental C9 onto E. coli and to enhance bacterial killing. The bactericidal activity of neonatal serum against multiple isolates of pathogenic E. coli was increased after C9 supplementation.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific induction of intracellular calcium oscillations by complement membrane attack on oligodendroglia.

Oligodendroglia (ODG) are unique among glial cell types in their capacity to activate complement in the absence of antibody, causing insertion of the potentially damaging membrane attack complex (MAC) into the plasma membrane. Using microfluorimetry of indo-1 fluorescence we have detected a complex oscillatory [Ca2+]i response in ODG following exposure to sublethal dilutions of serum-derived complement. Oscillations were transitory and preceded complete and stable return to resting [Ca2+]i levels, whereas nonoscillating ODG underwent rapid lysis. Depletion of the terminal complement component C9 from serum removed the oscillatory stimulus, which could be restored by reconstitution with purified C9. Exposure to the C9-homologous peptide melittin produced [Ca2+]i oscillations similar in pattern to those induced by whole serum. However, this type of response could not be reproduced by Ca2+ ionophores or mechanical wounding, suggesting that oscillations cannot be provoked by Ca2+ influx alone and depend on the presence of the MAC or a pore-forming lesion. Oscillations were not prevented in the continuous presence of caffeine, demonstrating independence from caffeine-releasable intracellular stores. Inhibition of the endoplasmic reticular Ca(2+)-ATPase with thapsigargin produced an abrupt elevation in [Ca2+]i but did not alter the latency between exposure to serum and the initial complement-induced transient. However, the slope of this initial transient was considerably reduced and oscillations suppressed, demonstrating dependence of the oscillatory mechanism on functional endoplasmic reticular Ca2+ stores. The coincidence of ODG recovery with oscillating [Ca2+]i suggests that the complex calcium signal that follows MAC attack may stimulate repair or protective mechanisms.

Molecular basis of complement resistance of human melanoma cells expressing the C3-cleaving membrane protease p65.

The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.

Inhibition of the complement membrane attack complex by the galactose-specific adhesion of Entamoeba histolytica.

The human complement system is an important early host defense against infection. Entamoeba histolytica activates the complement system but is resistant to killing by complement C5b-9 complexes deposited on the membrane surface. Our aim was to identify components of the amebic plasma membrane that mediate resistance to human complement C5b-9 by screening for neutralizing monoclonal antibodies. A monoclonal antibody was identified that abrogated amebic resistance to C5b-9, and the mAb was shown to recognize the parasite's galactose-specific adhesin. The purified adhesin bound to C8 and C9 and conferred C5b-9 resistance to sensitive ameba upon reconstitution; these activities of the adhesin were inhibited by the antiadhesin mAb. The E. histolytica adhesin shared sequence similarities and antigenic cross-reactivity with CD59, a membrane inhibitor of C5b-9 in human blood cells, suggesting both molecular mimicry and shared complement-inhibitory functions.

Membrane vesiculation protects erythrocytes from destruction by complement.

Nucleated cells can resist attack by C by exocytosis or endocytosis of the terminal C components C5b-9 (membrane attack complex) (MAC), but it is generally accepted that formation of a single MAC channel on E leads to lysis (one-hit theory). We find that human and guinea pig E, but not SRBC, can eliminate the MAC from the membrane in the form of microvesicles and escape destruction. When guinea pig or human E are incubated with C5b-9, vesiculation proceeds without a lag and is detected at nonlytic doses of C9. Continuous Ca2+ influx is required for vesiculation. The amount of released vesicles is in direct relation to Ca2+ concentration, and the increase in vesiculation is associated with a parallel decrease in lysis. SRBC, which do not vesiculate when Ca2+ loaded, are lysed by C5b-9 with the same efficiency in the presence or absence of Ca2+. Vesicles released from guinea pig RBC under C5b-9 attack are enriched in C9 by a factor of 10, compared with the unlysed cells, and by a factor of 3 to 4, compared with ghosts. We conclude that E are protected from lysis not only by CD59 and C8bp/HRF, which prevent MAC assembly, but also by selective elimination of the MAC.

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals.

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.

Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles.

During sublytic complement attack on human neutrophils, plasma-membrane vesicles are shed from the cell surface as a cell-protection mechanism. By using surface-iodinated neutrophils it was found that less than 2% of surface label was recovered in shed vesicles under conditions where 40% of complement component C9 was shed. SDS/PAGE of 125I-labelled shed vesicles and plasma membranes showed differences in iodination pattern, demonstrating the sorting of membrane proteins into the shed vesicles. Analysis of 32P-labelled phospholipids after labeling of neutrophils with [32P]Pi before sublytic complement attack showed the presence of phosphatidic acid, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and polyphosphoinositides in shed vesicles. Quantitative analysis using [3H]acetic anhydride-labelling method showed that the molar proportions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were the same in shed vesicles as in plasma membranes. In contrast, the molar proportions of cholesterol and diacylglycerol relative to sphingomyelin were almost twice those found in plasma membranes. The data demonstrate the existence of protein and lipid sorting mechanisms during the formation of shed vesicles when neutrophils are subject to sublytic complement attack. The term 'ectocytosis' is proposed to describe triggered shedding of right-side-out membrane vesicles from the surface of eukaryotic cells.

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites.

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.

Channel fluctuations induced by membrane attack complex C5B-9.

The assembly of complement (C) components C5b-9 in membranes results in the formation of transmembrane lesions. The C9 component has been shown to be mainly responsible for formation of the ultrastructurally visible tubules associated with C5b-9 complexes. Several studies have disputed the role of C9 polymerization in C-mediated cytolysis on the grounds that C5b-9 lyses cells in the absence of tubular formation. Here, C5b-9 complexes were reconstituted into high-impedance planar lipid bilayers and shown to form channels which are heterogenous in size. The smallest channels had unitary conductances of 15 picoSiemens (pS) in 0.1 M NaCl. The closing of these channels showed voltage-dependence at membrane potentials exceeding 40 mV. These channels were more cation-selective, with K+ ions being favored over Na+. The 15-pS channels described here are much smaller than the channels attributed previously to either C5b-9 or polymerized C9 complexes but resemble channels formed by the C9b fragment, which does not polymerize into tubules. These results indicate that C5b-9 complexes are capable of damaging membranes by forming initially small ion channels which then aggregate in the membrane to form tubular lesions with much larger conductances. Like C5b-9, C5b-8 also increased membrane permeability. However, this increase in membrane conductance could not be resolved into single channels, suggesting that C5b-8 may induce membrane leakiness by perturbing the packing of membrane lipids, whereas addition of C9 results in authentic production of ion channels.