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PubMed1987

Human complement factor I: analysis of cDNA-derived primary structure and assignment of its gene to chromosome 4.

Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.

Amino Acid Sequence↗
PubMed1985

Leukocyte complement: interleukin-like properties of factor Bb.

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".

Complement C3↗
PubMed1988

Differential effect of inflammatory stimuli on murine plasma C4 and factor B concentrations.

The in vivo effects of a variety of inflammatory stimuli on complement C4 and factor B plasma levels have been examined. MRL/++ (H-2k) mice were given intraperitoneal injections of lipopolysaccharide, turpentine, Corynebacterium parvum pyridine extract residue or high doses of indomethacin. All of these treatments induced an increase in plasma factor B concentrations, which in the case of C. parvum was dose dependent and persisted for at least 7 days. Lipopolysaccharide, turpentine and indomethacin produced decreases in plasma complement C4. C. parvum, however, produced an increase in plasma complement C4 to approximately 240% of controls which was independent of gender. It was also independent of major histocompatibility complex haplotype, since the same effect was seen in C57B1/6J-bg/bg and C57B1/6J-bg/+ mice. The gross increment in complement C4 was, however, related to the major histocompatibility complex. H-2K mice ("low complement C4") had smaller increments than H-2b ("high complement C4"). Mycobacterium bovis (BCG) also produced a transient increase in C4 in the H-2b mice as well as a prolonged increase in factor B levels. These data (i) suggest that different inflammatory stimuli induce different mediators which may have differential effects on factor B and complement C4 synthesis, and (ii) emphasize the independent regulation of complement C4 and factor B. Qualitative variations in the mediators elaborated during chronic inflammatory diseases may help determine complement C4 fluctuations in systemic lupus erythematosus and the wide range of complement C4 concentrations seen in MRL/1 pr mice with active immune complex disease.

Animals↗
PubMed1979

Properdin factor D: effects on thrombin-induced platelet aggregation.

Factor D, when preincubated with platelet suspensions, at concentrations as low as 1.2 micrograms/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor D were added to platelets at the same time. No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor D by increasing its concentration. Diisopropyl-phosphorofluoridate-inactivated factor D also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor D was not required for inhibition. Factor D absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 125I-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 micrograms/ml). Factor D is thus a competitive inhibitor of thrombin binding, although the affinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor D. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.

Binding, Competitive↗
PubMed1988

Solubilization of immune precipitates by complement in the absence of properdin or factor D.

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.

Antibodies↗
PubMed1992

Fatty acid synthase and adipsin mRNA levels in obese and lean JCR:LA-cp rats: effect of diet.

In Sprague-Dawley rats, fatty acid synthase (FAS) activity is suppressed by dietary fat. To test the hypothesis that a defect in regulation of de novo fatty acid synthesis exists in massive obesity, we investigated the effect of diet on FAS mRNA levels in genetically obese JCR:LA-corpulent (cp) rats. We also determined levels of mRNA encoding adipsin, a fat cell-derived protein possibly associated with lipid metabolism. Hepatic FAS mRNA levels were elevated five-fold in obese compared to lean cp rats and were unsuppressed by dietary fat. Dietary sucrose increased FAS mRNA levels in lean cp rats, but, in contrast to Sprague-Dawley rats, little deposition of lipid resulted. Adipsin mRNA levels were fivefold lower in obese cp and Sprague-Dawley rats than in lean cp rats and were unaffected by diet. We conclude that exaggerated de novo fatty acid synthesis may play a major role in the pathogenesis of obesity in obese JCR:LA-corpulent rats.

Adipose Tissue↗
PubMed1994

The adipsin-ASP pathway and regulation of adipocyte function.

The adipsin-ASP pathway provides a mechanism by which the adipocyte is able to regulate its rate of de novo triglyceride synthesis and reesterification. The adipocyte can synthesize and secrete the three proteins necessary for the formation of the effector molecule, acylation stimulating protein (ASP). ASP increases membrane transport of glucose and the activity of diacylglycerol acyltransferase and by virtue of both of these effects markedly increases the rate of triglyceride synthesis. Awareness of the pathway will allow, we believe, a new understanding of the regulation of triglyceride removal from plasma. Accordingly, the concept of microenvironmental metabolic regulation of triglyceride hydrolysis at the endothelial surface and triglyceride synthesis within the adipocyte will be presented. In addition, the pathogenetic sequence by which dysfunction of this pathway can lead to dyslipoproteinaemia will be reviewed. Emphasis, however, will be placed on the role this pathway may play in the pathogenesis of obesity and the adaptation to negative caloric balance in the obese.

Adipocytes↗
PubMed1980

Active site amino acid sequence of human factor D.

Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

Amino Acid Sequence↗
PubMed1990

Combined lipase deficiency in the mouse. Evidence of impaired lipase processing and secretion.

Newborn combined lipase-deficient (cld) mice have severe hypertriglyceridemia associated with a marked decrease of lipoprotein lipase (LPL) and hepatic lipase (HL) activities. Since the cld mutation and lipase genes reside on separate chromosomes, combined lipase deficiency cannot result from defects occurring within the LPL or HL structural genes. To elucidate the biochemical basis of this trans-acting defect, cld mice were compared to unaffected littermates for changes in lipase mRNA levels, rates of synthesis, and posttranslational processing and secretion. LPL and HL mRNA levels in cld liver and LPL in cld heart were comparable to controls; corresponding lipase synthetic rates were modestly decreased by about 30%. However, these reduced synthetic rates were not lipase-specific, since the rates of apolipoprotein (apo) A-I and apoA-II synthesis in cld liver were similarly decreased. Despite LPL synthetic rates that were 70% of controls, LPL mass in cld postheparin plasma was markedly reduced to only 7% of control values, suggesting that the majority of LPL is not secreted but remains intracellular. Consistent with a lipase secretory defect, neither the LPL nor HL oligomannosyl forms were converted to their respective complex forms in cld tissues, indicating that the lipases had failed to move from the endoplasmic reticulum/cis-Golgi to the medial/trans-Golgi network. In addition, the majority of intracellular LPL was catalytically inactive, since LPL specific activity (units/mg LPL protein) in cld heart, kidney, and brain was reduced 80-97%. In contrast to the severe impairment of lipase posttranslational processing and secretion, cld mouse plasma contained normal levels of another secretory N-linked glycoprotein, adipsin, with its oligosaccharide chains fully processed to the complex form. Thus, the cld mutation appears not to globally disrupt the secretion of all N-linked glycoproteins, but rather selectively impairs LPL and HL at points essential to their normal intracellular transport and secretion.

Animals↗
PubMed1984

Isolation and partial characterization of bovine and equine factor D.

Bovine and equine factor D were purified to apparent homogeneity as evidenced by a single protein staining band on 7.5-17.5% SDS-PAGE slab gels under both reducing and non-reducing conditions. An apparent mol. wt of 15,000 for bovine D and 22,500 for equine D were noted after SDS-PAGE gel analysis of both reduced and non-reduced preparations. A single polypeptide chain for both proteins was evidenced by the lack of any change in the electrophoretic mobility under each of these conditions. The bovine and equine D were enriched 3347- and 9447-fold, with a 20 and 29% yield of hemolytic activity, respectively. Functionally, both equine and bovine D would reconstitute a human reagent deficient in D (RD), while human or equine D would substitute for bovine D when using a bovine RD. Neither bovine, equine or human D would, however, reconstitute an equine RD.

Animals↗
PubMed1989

Adrenal glucocorticoids regulate adipsin gene expression in genetically obese mice.

Adipsin expression at the protein and mRNA levels is greatly reduced in several distinct syndromes of obesity in the mouse: genetic obesity due to the db/db and ob/ob genes, and a chemically induced model secondary to neonatal exposure to monosodium glutamate. We considered first the possibility that the adipsin gene might be identical to the db or ob locus and the lowered expression of this protein might result from a mutation in this gene. We show here that the adipsin structural gene is located on chromosome 10 and hence is physically distinct from any obesity genes so far identified in the mouse. A major role for the adrenal gland and adrenal glucocorticoids in the aberrant regulation of adipsin in these models of obesity is indicated by several experiments. Adrenalectomy of the ob/ob mouse raises the circulating levels of adipsin protein and the amount of this mRNA in epididymal fat pads (5-fold), although neither is increased to the levels seen in lean controls. Exogenous administration of corticosterone completely blocks the effects of adrenalectomy on adipsin, suggesting that the effect of this endocrine ablation is through reduction of adrenal glucocorticoids. Corticosterone administration also causes suppression in the levels of adipsin mRNA and protein in lean mice, although this decrease is never as severe as that seen in obese mice. The effect of exogenous corticosterone in lean mice occurs within 2 days and hence is not secondary to the obesity which these hormones eventually elicit. These results indicate that glucocorticoids can regulate adipsin expression in vivo and strongly suggest that the hyperglucocorticoid state seen in certain obese models plays a significant role in lowering adipsin mRNA and protein levels. Quantitative analysis of these experiments suggests that other as yet unknown neuroendocrine factors also function to suppress adipsin in obesity.

Adipose Tissue↗
PubMed1983

Effect of cycloheximide and of anti-C3 Fab' on the intrinsic synthesis and secretion of lysosomal enzyme and of complement components by guinea-pig peritoneal macrophages.

Without any additional stimulus, the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (beta-GLU) is secreted immediately from starch gel induced guinea-pig peritoneal macrophages. A more than three-fold increase of the total enzyme activity in the course of 3 days and the reversible inhibition of this enzyme increase by cycloheximide prove the synthesis of beta-GLU. The synthesis rate of beta-GLU remains rather constant after the second day, despite rapid cell death in the same culture period which could be explained by heterogeneity of the macrophage population. Cycloheximide immediately inhibits secretion of C3 whereas inhibition of beta-GLU secretion is only observed after a lag phase of 24 h. Secretion of factor D and of beta-GLU is not altered if secreted C3 is totally neutralized by the addition of anti-C3 Fab' to macrophage cultures. Thus, endogenous C3 as well as its endogenously generated fragments do not influence these macrophage functions. In the same cultures with anti-C3 Fab', conversion of secreted factor B into its fragments is inhibited as indicated by the detection of functional B activity. These results indicate that the secretion of factor D and of beta-GLU is also independent of endogenous B-derived fragments such as Bb. Finally, the detection of functional B in cultures with anti-C3 Fab' proves that C3b is required for factor B activation. The apparent interaction of secreted C3 and factors D and B in regular macrophage cultures suggests the constant formation of the labile C3 convertase C3bBb of the alternative pathway.

Acetylglucosaminidase↗
PubMed1983

Human factor D of the alternative pathway: purification and quantitation by enzyme amplified electroimmunoassay.

Purified factor D was prepared with a yield of about 30%. Monospecific antiserum was raised in rabbits. Immunochemical quantitation of factor D in serum and plasma was performed by electroimmunoassay and a sensitive staining technique based on enzyme amplification. Peroxidase-labeled swine antibodies to rabbit immunoglobulin were applied to the gel after electrophoresis. Immune precipitates were then visualized by staining for peroxidase activity. Factor D could be detected at 50 micrograms/l. The normal concentration of D in serum was 1.6 mg/l, as assessed by this assay.

Animals↗
PubMed1991

Purification and partial characterization of rat factor D.

Rat factor D has been purified to homogeneity (10,559-fold) from serum by chromatography on CM-Sepharose Fast Flow, phenyl-Sepharose CL-4B and Mono S and has been shown to resemble its human and mouse counterparts both in substrate specificity and in its susceptibility to inhibition by the organophosphorous inhibitor di-isopropylfluorophosphate. The rat enzyme, however, is heavily glycosylated and binds to wheat-germ lectin-Sepharose 6MB and 5-hydroxytryptamine-agarose, but not to concanavalin A-Sepharose 4B. All of the carbohydrate chains are N-linked. Enzymic removal of this carbohydrate decreased the Mr by approx. 15,000. The deglycosylated rat enzyme had the same mobility as native human factor D on SDS/PAGE, corresponding to an Mr of 24,500. N-Terminal sequence analysis of the first 30 amino acids of rat factor D highlighted the sequence similarity with human factor D (greater than 76%) and, in particular, with mouse adipsin (greater than 93%).

Amino Acid Sequence↗
PubMed1987

Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-fos.

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including a putative lipid-binding protein termed adipocyte P2 (aP2). The aP2 gene contains a regulatory element (FSE2) 124 bases 5' to its start of transcription. This element binds nuclear factors in sequence-specific and differentiation-dependent fashion as determined by altered mobility in gel retardation assays. Deletion analysis of promoter-linked transfection assays and competition of these constructions in cells with a synthetic FSE2 element suggest that trans-acting factors bind to this region and act as negative regulators of aP2 gene activity in preadipocytes. c-fos appears to participate directly in this nucleoprotein complex, as demonstrated by the ability of antibodies to c-fos to disrupt specific binding of factors to the FSE2 sequence but not to factor-binding sequences from several other genes. Antibodies to c-fos specifically immunoprecipitate protein complexes covalently bound to FSE2 DNA via UV cross-linking.

Adipose Tissue↗
PubMed1980

Partial amino acid sequence of human factor D:homology with serine proteases.

Human factor D purified to homogeneity by a modified procedure was subjected to NH2-terminal amino acid sequence analysis by using a modified automated Beckman sequencer. We identified 48 of the first 57 NH2-terminal amino acids in a single sequencer run, using microgram quantities of factor D. The deduced amino acid sequence represents approximately 25% of the primary structure of factor D. This extended NH2-terminal amino acid sequence of factor D was compared to that of other trypsin-related serine proteases. By visual inspection, strong homologies (33--50% identity) were observed with all the serine proteases included in the comparison. Interestingly, factor D showed a higher degree of homology to serine proteases of pancreatic origin than to those of serum origin.

Amino Acid Sequence↗
PubMed1985

In vitro biosynthesis of complement protein D by U937 cells.

Preliminary studies demonstrating the secretion of antigenic D by blood monocytes/macrophages led us to study the biosynthesis of D by U937 cells, a human monocyte cell line. The kinetics of secretion of D into cell culture supernatants were followed by a solid-phase radioimmunoassay and by hemolytic assay. Daily synthesis of antigenic D was nearly linear (mean +/- 1 SD = 5.3 +/- 2.2 ng D/10(6) cells) over a 6-day period. The D produced after day 2 was hemolytically active, with a specific hemolytic activity greater than (although in the same range as) D in normal serum. Cycloheximide (10(-7) M) inhibited D synthesis, which returned to the levels found in untreated cells after removal of the inhibitor. Supernatants and lysates of cells grown in the presence of [35S]methionine were incubated with rabbit anti-D serum or FD10-1, a monoclonal anti-D antibody, bound to protein A-agarose. Autoradiograms of SDS-PAGE analysis of the precipitates demonstrated a main band of an approximate m.w. of 24,000, co-migrating with purified 125I-D. Identity of this band with D was established by blocking with excess purified D. Pulse-chase studies with the use of [35S]cysteine demonstrated a single D band both intra and extracellularly. Both forms of D had the same apparent m.w. which was approximately 3000 heavier than control 125I-D. These data demonstrate that U937 cells synthesize functionally active D, which appears to be structurally and antigenically similar to D in serum.

Amino Acids↗