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At least 19 recordsLinked to original sources
PubMed1989

Adipsin and complement factor D activity: an immune-related defect in obesity.

Adipsin is a serine protease that is secreted by adipocytes into the bloodstream; it is deficient in several animal models of obesity, representing a striking example of defective gene expression in this disorder. Recombinant mouse adipsin was purified and its biochemical and enzymatic properties were studied in order to elucidate the function of this protein. Activated adipsin has little or no proteolytic activity toward most substrates but has the same activity as human complement factor D, cleaving complement factor B when it is complexed with activated complement component C3. Like authentic factor D, adipsin can activate the alternative pathway of complement, resulting in red blood cell lysis. Decreased (58 to 80 percent) complement factor D activity, relative to lean controls, was observed as a common feature of several experimental models of obesity, including the ob/ob, db/db, and monosodium glutamate (MSG)-injected mouse and the fa/fa rat. These results suggest that adipsin and the alternative pathway of complement may play an unexpected but important role in the regulation of systemic energy balance in vivo.

Adipose Tissue↗
PubMed1992

Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue.

A cDNA for human adipsin was isolated and shown to encode a protein sharing 98% amino acid sequence similarity with the protein sequence previously determined for purified natural human complement factor D. Like mouse adipsin, recombinant human adipsin displays the enzymatic activity of human complement factor D, cleaving complement factor B only when B is complexed with activated complement component C3. We conclude that human adipsin is equivalent to complement factor D and that adipsin is the homologue of factor D in rodents. Adipose tissue is a major site of synthesis of human adipsin/complement factor D mRNA, but unlike the case in rodents, human adipsin mRNA is also expressed in monocytes/macrophages. The data presented here, demonstrating the equivalence of human adipsin to complement factor D and its high level of expression in fat, suggest a previously unsuspected role for adipose tissue in immune system biology.

Adipose Tissue↗
PubMed1989

Complete and partial deficiencies of complement factor D in a Dutch family.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.

Adult↗
PubMed1990

A monoclonal antibody which blocks the function of factor D of human complement.

Factor D is an essential enzyme for activation of complement by the alternative pathway (AP). It has been difficult to obtain mouse monoclonal antibodies (Mabs) which block the function of factor D. We have developed a strategy to obtain such Mabs using a double screening procedure of the initial clones. We selected the clone whose supernatant had the lowest level of anti-factor D Ab by ELISA and abolished factor D haemolytic activity. Addition of this Mab to human serum was shown to abolish conversion of C3 by cobra venom factor, haemolysis of rabbit erythrocytes, and activation of C3 and C5 by cuprophane dialysis membranes.

Animals↗
PubMed1991

Purification of human complement factor D from the peritoneal fluid of patients on chronic ambulatory peritoneal dialysis.

We describe a simple procedure for the purification of human factor D from the peritoneal fluid (PF) of patients with end stage renal failure (ESRF) on chronic ambulatory peritoneal dialysis (CAPD). The main advantages of this method are: (1) a relative enrichment of factor D in PF versus plasma (factor D/total protein enriched 3.8-fold); thus, added to the elevated concentration of factor D in ESRF, the enrichment compared to normal human serum is approximately 50 fold. (2) This biological source of factor D is almost unlimited, since around 10 liters of PF are removed per day from each patient. The purification is performed in three simple steps-Bio Rex 70, Heparin Sepharose CL 6B and Mono S FPLC- and milligrams of highly purified factor D are obtained. Peritoneal fluid might be a valuable source for the purification of other low MW proteins which accumulate in renal failure.

Antibodies, Monoclonal↗
PubMed1988

Metabolism of complement factor D in renal failure.

Factor D is an essential enzyme of the alternative pathway of complement. Its plasma concentration increases approximately tenfold in end-stage renal failure (ESRF). To analyze its metabolism in humans, we injected purified radiolabelled factor D into 5 healthy individuals and 12 patients with various renal diseases or renal failure. Fractional metabolic rates (FMR) and extravascular/intravascular distributions (EV/IV) were calculated using a compartmental model. The FMR was very rapid in normal individuals (mean 59.6%/hr; range 74.1 to 50.5), significantly diminished in the five patients with ESRF (5.7%/hr; 7.0 to 2.8; P less than 0.004), and correlated well with the creatinine clearance (r = 0.89; P less than 0.001). The extrarenal catabolic rate was not modified in renal failure. Despite a significant inverse correlation between plasma levels of factor D and creatinine clearance [r = 0.68; P less than 0.002], factor D levels were not a sensitive indicator of renal function because the synthesis rate (SR) varied widely from one individual to another (mean SR: 62.9 micrograms/kg/hr; 14.9 to 136.5). Factor D synthesis was not significantly altered by renal function, and did not correlate with C-reactive protein, suggesting that factor D is not an acute phase protein. The proportion of intact factor D elimination in the urine was increased in patients with tubular dysfunction (up to 15% compared to less than 0.2% in normal individuals) confirming that under normal circumstances factor D is filtered through the glomerulus and catabolized by tubular cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement Activating Enzymes↗
PubMed1991

[Factor D of complement: Uremic toxin?].

Factor D is an essential enzyme of the alternative pathway of complement. Its catabolism is mainly renal. The concentration of factor D increases with renal failure, and is approximately 10-fold higher in patients with end-stage renal disease. The accumulation of factor D is responsible for an enhancement of alternative pathway activation. Whether this excess of factor D has pathophysiological consequences remains to be determined, however, complement activation might participate in adverse effects during hemodialysis and in the progression of renal injury.

Complement Factor D↗
PubMed1991

Crystallization and preliminary X-ray investigation of factor D of human complement.

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 A.

Complement Factor D↗
PubMed1978

Mechanism of action of factor D of the alternative complement pathway.

Factor D (C3 proactivator convertase) of human serum has been shown to be absolutely necessary for alternative pathway function, for activation of the C3/C5 convertase of that pathway and not to be a subunit of this enzyme. Factor D was found to be present in human plasma in active form only, at a concentration of 2 microgram/ml, and not to be controlled by plasma protease inhibitors or by spontaneous decay. Unlike trypsin, factor D cleaves and activates factor B only when it is in Mg++-dependent complex with C3b, has no esterolytic activity, and is unable to cleave the B chain of insulin. The alleged functional and antigenic relationship of factor D to alpha-thrombin could not be verified. The results of this study led to the description of the mechanism of action of factor D in terms of the cryptic site hypothesis.

Complement Activating Enzymes↗
PubMed1991

Complement factor D-like activity of Porphyromonas gingivalis W83.

Porphyromonas gingivalis is a proteolytic gram-negative anaerobic bacterium that is frequently isolated from lesions of human periodontal disease. Previous studies have shown that P. gingivalis strain W83 inactivates C3 in pooled normal human serum (NHS) by a mechanism that is inhibitable by EDTA, yet it degrades purified complement proteins by a mechanism that is not EDTA-inhibitable. Furthermore, during complement activation, only a small number of C3 molecules accumulate on the surface of this organism unless the bacteria are treated with the protease inhibitor TLCK prior to complement activation. The hypothesis was tested that P. gingivalis W83 contains protease activity mimicking that of complement factor D, thus enabling it to activate C3 in serum without significant C3 accumulation on the cell surface. It was first noted that incubation of P. gingivalis W83 in absorbed human serum that was depleted of factor D resulted in C3 consumption that was reversed in the presence of the protease inhibitor TLCK. To directly demonstrate that factor B-dependent C3 consumption occurs in the absence of factor D, P. gingivalis W83 was incubated with purified C3 or a mixture of C3 and B. Although some proteolysis of C3 was noted, increased C3 consumption was noted in mixtures containing both C3 and B. This increment in C3 consumption was inhibited by both EDTA and TLCK. Furthermore, the addition of purified factor H to this mixture inhibited the increment in C3 consumption, indicating that a C3 convertase was probably formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Gel↗
PubMed1989

Regulation of the human alternative complement pathway: formation of a ternary complex between factor H, surface-bound C3b and chemical groups on nonactivating surfaces.

Sephadex [alpha(1----6) cross-linked dextran] activates the human alternative pathway of complement. Substitution of hydroxyl groups of Sephadex with carboxymethyl groups (CM) results in a dose-dependent decrease of the activating capacity of the polymer in normal human serum. Sephadex bearing one CM group/glycosyl unit (CM-Seph 0.95) exhibited no activating capacity. CM groups did not interfere with the ability of the polymer to covalently bind C3b in the presence of purified alternative pathway proteins nor with the capacity of bound-C3b to form a C3 convertase in the absence of regulatory proteins. C3b that was bound to CM-Seph 0.95 was more susceptible to inactivation by factors H and I in serum than C3b bound to Sephadex. Binding studies using 125I-labeled H demonstrated that H bound with a similar affinity to the activating particle Sephadex, to Sephadex bearing C3b and to the nonactivating particle CM-Seph 0.95. However, factor H bound with a 5- to 7-fold higher affinity to CM-Seph 0.95 bearing C3b. These results demonstrate a requirement for both CM groups and C3b molecules in order for H to bind with high affinity to C3b on the non-activating surface, and indicate that H formed a ternary complex with surface-bound C3b and CM groups on CM-Seph 0.95. Using a chemically defined model system, the present study provides a molecular basis for the enhanced interaction between surface-bound C3b and factor H on nonactivators of the human alternative pathway.

Complement Activation↗
PubMed1990

Molecular and functional identification and purification of complement component factor D from urine of patients with chronic renal failure.

Urine proteins of normal subject and patients with impaired renal function were analyzed by two-dimensional polyacrylamide gel electrophoresis. As a result, a clear spot was detected specifically in urine from patients with obvious renal dysfunction. The isoelectrical point of this unique spot was pH 7.1-7.2 and the flow-rate (Rf) was 0.50-0.55 as that of albumin was 1.0. Partial amino acid sequence analysis revealed that the NH2-terminal to 22nd amino acid sequence was identical with that of complement factor D. We purified 22 mg of this protein (factor D) from 5000 ml of urine from a patient on hemodialysis by three chromatographic steps using DEAE-Sephadex A-50 and Sephacryl S-200. The purified urine factor D gave a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis at the position of 23 kD, and displayed normal factor D hemolytic activity. The concentrations of factor D estimated by hemolytic assay were 1.9 micrograms/ml of normal serum, less than 0.1 microgram/ml of normal urine, 15 micrograms/ml of patient serum and 50 micrograms/ml of patient urine.

Amino Acid Sequence↗
PubMed1991

Deterioration of immune complex solubilization activity of serum by increased concentration of factor D.

We investigated the effect of excess complement factor D on the immune complex solubilization activity (ICSA) of serum. First, we estimated the concentration of factor D, ICSA and the hemolytic activity via the classical complement pathway (CH50) in the sera of 16 healthy individuals and 36 patients on hemodialysis for end-stage renal failure. The serum concentration of factor D in these patients (mean +/- SD: 12.12 +/- 2.38 micrograms/ml) was significantly higher (p less than 0.001) than that in the healthy subjects (1.02 +/- 0.11 micrograms/ml). In this study, peroxidase and antiperoxidase rabbit IgG were used as immune precipitates for ICSA. The ICSA in patients (45.8 +/- 7.4 normal human serum %, NHS%) was significantly lower (p less than 0.001) than that in the healthy subjects (100.2 +/- 12.5 NHS%). There was no difference in CH50 between the sera of the patients (31.8 +/- 2.5) and that of the healthy group (32.4 +/- 2.6). Second, we determined that increasing amounts of purified factor D added to fresh serum resulted in a decrease in ICSA. This occurred in the serum of a healthy individual as well as in that of a patient. CH50 did not change regardless of the concentration of factor D used. There was an increase in C3 conversion in the sera to which purified factor D had been added, as observed by crossed immunoelectrophoresis. It is suggested that ICSA had deteriorated due to the excess of factor D, which had activated the alternative pathway of complement.

Antigen-Antibody Complex↗
PubMed1990

Quantitation of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays.

Sensitive enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies have been developed to specifically detect components of the alternative pathway of complement in human blood plasma. Normal values of the factor B split products Ba (1.01 +/- 0.30 micrograms/ml, mean +/- SD), Bb (0.65 +/- 0.23 micrograms/ml), of the C3-fragments C3b/iC3b/C3dg (17.9 +/- 5.7 micrograms/ml), native factor B (238 +/- 48 micrograms/ml), factor D (1.05 +/- 0.27 micrograms/ml), and factor H (702 +/- 292 micrograms/ml) were determined in the EDTA-plasma of healthy probands (n = 55). The simultaneous quantitation of the main cleavage products and of control proteins in the plasma samples permits precise analysis of the activation of the alternative pathway of complement in various disease states. In addition, we describe a method for the specific depletion of factor B prior to fragment-specific assays utilizing monoclonal antibodies conjugated to paramagnetic beads. The latter should permit the quantitation of other complement split products.

Animals↗
PubMed1986

Alternative complement pathway activation by C4b deposited during classical pathway activation.

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.

Animals↗