Complement Factor H Is a Serum-binding Protein for Adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners*

Rubén PíoDagger §, Alfredo MartínezDagger , Edward J. Unsworth, Jeffrey A. Kowalak, José A. Bengoechea||, Peter F. Zipfel**, Ted H. ElsasserDagger Dagger , and Frank CuttittaDagger

From the Dagger  Department of Cell and Cancer Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, the  Laboratory of Neurotoxicology, National Institute of Mental Health, Bethesda, Maryland 20892, the || Department of Medical Biochemistry, Turku University, 20520 Turku, Finland, the ** Department of Infection Biology, Hans Knoell Institute for Natural Products Research, 07745 Jena, Germany, and the Dagger Dagger  Growth Biology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705

Received for publication, August 28, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human adrenomedullin (AM)1 is a 52-amino acid peptide originally isolated from a human pheochromocytoma and identified as a molecule capable of elevating rat platelet cAMP (1). AM belongs to the calcitonin gene peptide superfamily based on its slight homology with calcitonin gene-related peptide (CGRP) and amylin (1). The human mRNA is 1.6 kilobases long and encodes for a predicted 185-amino acid precursor from which two amidated peptides are generated: AM and a second peptide denoted as proadrenomedullin N-terminal 20 peptide (PAMP) (2). The expression of AM has been demonstrated in many tissues and biological fluids such as plasma (3), cerebrospinal fluid (4), sweat (5), amniotic fluid (6), urine (7), and milk (8). AM has been implicated in the modulation of numerous physiological functions, which include cardiovascular tone, central brain activity, bronchodilation, renal function, hormone secretion, cell growth, differentiation, and immune response (9).

Recently, we have demonstrated that plasma proteins from several species can specifically bind AM (10). The existence of these binding proteins was established using a radioligand blotting technique based on a method originally described for the detection of insulin-like growth factor-binding proteins (11). Most of the species analyzed, including humans, had an AM-binding protein (AMBP) with a Mr of 120,000 under nonreducing conditions. Interestingly, the plasma from ruminant species (calf, goat, and sheep) had an additional band of Mr 140,000. Whether these proteins are different or represent two glycosylation patterns from the same protein remains to be determined. An analysis of plasma from calves undergoing an acute phase response to a parasitic infection revealed a decrease in the expression of AMBP as compared with uninfected calves (10), whereas AM levels increased (12). The presence of a protein that specifically binds AM and the regulation of its expression in pathological situations may have a critical impact on AM physiology.

In this study, we isolate and characterize the human AMBP (AMBP-1) from plasma as complement factor H. We also describe how factor H may interfere with the quantification of AM by conventional radioimmunoassay (RIA) and how both binding partner proteins may modulate their respective biological activities.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Labeling of Adrenomedullin-- Nonradioactive labeling was accomplished by conjugation of synthetic AM-(1-52) (Peninsula Laboratories, Belmont, CA) with fluorescein-5-EX succinimidyl ester (Molecular Probes, Inc., Eugene, OR). Briefly, 100 µg of AM were dissolved in 1 ml of 50 mM sodium bicarbonate, pH 8.5, and the succinimidyl ester was added to a final molar concentration ratio of 10:1 (linker:AM). The mixture was incubated with slow agitation for 1 h at room temperature, and the reaction was terminated by the addition of 0.1 M ethanolamine, pH 8.5, followed by another incubation of 1 h. Labeled AM was mixed with an equal volume of 0.1% alkali-treated casein (ATC) in Tris-buffered saline (TBS), pH 7.4, and extracted using reverse phase Sep-Pak C18 cartridges (Waters, Milford, MA) with 80% acidic isopropyl alcohol as the elution buffer. The extract was lyophilized and reconstituted in 1 ml of TBS containing 0.1% ATC, 0.1% Tween 20, and 0.05% Triton X-100, pH 7.4. Fluorescein-labeled AM was stored at 4 °C for as long as 3 months without significant loss of activity.

Nonradioactive Ligand Blotting Assay-- Proteins from human plasma (2 µl) were electrophoretically fractionated on 3-8% Tris acetate gels (Novex, San Diego, CA) under nonreducing conditions and transferred to a 0.2-µm nitrocellulose membrane. The membrane was washed with 1% Nonidet P-40, blocked with 0.1% ATC in TBS, and incubated with 50 nM fluorescein-labeled AM for 16 h at 4 °C in blocking buffer containing 0.1% Tween 20. Binding was detected with a primary rabbit anti-fluorescein IgG (1:1000; Molecular Probes, Inc.), a secondary antibody coupled to alkaline phosphatase (1:2000; Dako, Carpinteria, CA), and nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Roche Molecular Biochemicals) as the color-substrate solution. For competition studies with unlabeled peptides, the membrane was preincubated with 5 µM unlabeled ligands at 4 °C for 6 h. Then labeled AM was added, and the membrane was incubated for 16 h at 4 °C. AM fragments, PAMP, and CGRP were purchased from Phoenix Pharmaceuticals (Belmont, CA).

Reverse-phase HPLC-- Preparative reverse phase HPLC was performed using a Delta Pak C18 300-Å column (30 mm × 30 cm; Waters, Tokyo, Japan) and the "System Gold" modular system (Beckman Instruments Inc., Fullerton, CA). 2.5 ml of human plasma were mixed with an equal volume of 10% acetonitrile with 0.2% trifluoroacetic acid, processed through a 0.2-µm filter, and loaded onto the column. After 5 min with 0.1% trifluoroacetic acid in 5% acetonitrile, the column was eluted with a linear gradient of acetonitrile containing 0.075% trifluoroacetic acid from 5 to 60% at a flow rate of 12 ml/min over 60 min. Each fraction (12 ml) was collected, freeze-dried, and dissolved in 0.3 ml of TBS, 0.1% Tween 20. Fractions were tested for the presence of AMBP-1 using the nonradioactive ligand blotting technique.

Amino Acid Analysis-- Amino acid analysis was performed by The Protein/DNA Technology Center at the Rockefeller University, New York. HPLC (NovaPak C18 30-cm column) with the Waters PicoTag Work station and a two-pump gradient system (model 510) equipped with a model 490 UV multiwavelength detector were used as previously described (13).

Edman Degradation-- The N-terminal sequence analysis was performed by the Biotechnology Resource Laboratory, Protein Sequencing and Peptide Synthesis Facility (Medical University of South Carolina, Charleston, SC). The sample was subjected to automated Edman degradation using a PE Biosystems Procise 494 Protein Sequencer and a PE Biosystems cLC Microblotter 173, using standard cycles and reagents (14, 15).

Mass Spectrometry-- After fractionation by SDS-polyacrylamide gel electrophoresis under reducing conditions (5% beta -mercaptoethanol), the gel was stained with Coomassie Blue, and the AMBP-1 band was excised. In-gel protein digestion and peptide extraction were performed as previously described (16). One-tenth of the extracted protein digest was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on a PerSeptive Voyager-DE STR (PE Biosystems, Foster City, CA) prior to liquid chromatography/mass spectrometry. The instrument was operated in reflector mode with the accelerating voltage set to 20,000, the laser energy to 2350, the guide wire voltage to 0.05%, and the grid voltage to 95%. The mirror ratio was set to 1:110. The remainder of the extracted protein digest was injected onto a 0.3 × 100-mm, 5-µm BetaBasic C18 column (Keystone Scientific, Bellafonte, PA), which had been equilibrated with 10% buffer B in buffer A (A: water with 0.1% formic acid; B: acetonitrile with 10% 1-propanol and 0.1% formic acid). Peptide elution was carried out using a linear gradient progressing from 10 to 60% buffer B over 60 min (Shimadzu Scientific Instruments LC10AD/VP pumps and LC10A controller). The eluting peptides were detected by a Finnigan LCQ mass spectrometer (ThermoQuest; Finnigan MAT Division, Piscataway, NJ). Peptide sequence data were obtained from the eluting peptides by MS/MS on those ions exceeding a preset threshold of 5 × 104 ions. The operating parameters were as follows: sheath gas flow, = 32, auxiliary gas flow = 1, spray voltage = 4.5 kV, capillary temperature = 200 °C, capillary voltage = 8.0 V, and tube lens offset = -20 V.

Western Blot-- Proteins were electrophoretically fractionated on a 3-8% Tris acetate (for factor H) or a 4-12% Bis-Tris gel (for AM) under nonreducing conditions, transferred to a 0.2-µm nitrocellulose membrane, and blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS). Afterward, the membrane was incubated with 1:2000 anti-factor H rabbit antibody (Quidel, San Diego, CA) or 1:4000 anti-AM rabbit antibody (17) and developed using the ECL Plus Western Blot Detection System (Amersham Pharmacia Biotech).

Binding Assay-- A 96-well polyvinyl chloride plate was coated with factor H (5 ng/well; Sigma). The plate was blocked (TBS, 0.1% ATC, 0.1% Tween 20) and incubated with the unlabeled peptides for 2 h. Fluorescein-labeled AM (50 nM) was added, and after a 2-h incubation the assay was developed using an anti-fluorescein polyclonal antibody (1:1000, Molecular Probes, Inc.) and 125I-Protein A (Amersham Pharmacia Biotech). The radioactivity was determined in a gamma -counter.

AM Immunoprecipitation-- 3 ml of sample were mixed with 1 ml of Protein A-agarose (Life Technologies, Inc.) containing a 1 µM final concentration of each of the following protease inhibitors: pefabloc (Centerchem Inc., Stamford, CT), bestatin, and phosphoramidon (Sigma). After 1 h at 4 °C, the sample was centrifuged, and the supernatant was divided and further incubated with 80 µl of rabbit anti-AM (17) or rabbit preimmune serum for 1 h at 4 °C. Protein A-agarose (80 µl) was then added to the mix. After a 30-min incubation, the immunoprecipitate was collected by centrifugation, and the pellet was extensively washed with TBS, 0.1% Triton X-100. The final pellet was resuspended in 100 µl of LDS sample buffer (Novex) and boiled before the Western blot analysis.

Extraction of Plasma and AM Radioimmunoassay-- Extraction was performed using reverse-phase Sep-Pak C18 cartridges (Waters) as previously reported (18, 19). Briefly, cartridges were activated with 80% methanol and washed with 0.9% NaCl. Plasma samples were mixed with an equal volume of PBS containing 0.1% ATC and 0.1% Triton X-100, pH 7.4. Samples were applied to the columns, and, after washing twice with 0.9% NaCl, AM was eluted with 80% isopropyl alcohol containing 125 mM HCl. Extracts were freeze-dried to remove the organic solvent. Concentrations of AM in the extracts were measured by radioimmunoassay as previously described (19).

cAMP Assay-- Rat-2 fibroblasts were grown in RPMI 1640 containing 10% fetal bovine serum (Life Technologies). Cells were seeded into 24-well plates at 2 × 104 cells/well and incubated 48 h at 37 °C in 5% CO2. Before the assay, cells were incubated in TIS medium (RPMI 1640 plus 10 µg/ml transferrin, 10 µg/ml insulin, and 50 nM sodium selenite) for 15 min. Then cells were treated for 5 min with AM (Bachem, King of Prussia, PA) and/or factor H (Sigma) in 250 µl of TIS medium containing 1% BSA, 1 mg/ml bacitracin, and 100 µM isobutylmethylxanthine. The reaction was terminated by adding an equal volume of ice-cold ethanol. cAMP was measured using the Biotrac cAMP RIA (Amersham Pharmacia Biotech).

Receptor Binding Assay-- Binding of rat 125I-AM (Phoenix Pharmaceuticals) to Rat-2 cells was determined as previously reported (20). In brief, Rat-2 cells were plated out at 2 × 104 cells/well in poly-D-lysine-coated 24-well plates (Becton Dickinson, Bedford, MA). Confluent cells were incubated for 60 min at 4 °C in 0.5 ml of binding buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 10 mM NaCl, 4 mM KCl, 1 mM EDTA, 1 µM phosphoramidon, 0.25 mg/ml bacitracin, 0.3% bovine serum albumin) containing 100 pM rat 125I-AM. After incubation, cells were washed twice with ice-cold binding buffer and then dissolved in 1 M NaOH for counting. Nonspecific binding was measured by incubating the cells with a 1000-fold excess of unlabeled AM.

Proliferation Assay-- The breast cancer cell line T-47D (ATCC, Manassas, VA) was maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies). The MTT Proliferation Assay (Promega, Madison, WI) was carried out in serum-free conditions as previously reported (21). Briefly, cells were seeded in 96-well plates at 1-2 × 104 cells/well, and appropriate concentrations of the indicated compounds were added. After 5 days of incubation at 37 °C and 5% CO2 in a humid incubator, the MTT colorimetric assay was carried out following the instructions from the manufacturer. The plate was read at a wavelength of 540 nm. Eight independent wells per treatment were averaged.

Antimicrobial Activity Assay-- The antimicrobial activity was measured using Escherichia coli (ATCC 35218, Gaithersburg, MD) and a radial diffusion assay as previously described (22). Briefly, bacteria were incorporated into a thin underlay gel that contained 1% agarose, 2 mM HEPES, pH 7.2, and 0.3 mg/ml of trypticase soy broth powder. After polymerization, small wells of 10 µl of capacity were carved in the agar. Test substances were added and allowed to diffuse for 3 h at 37 °C. A 10-ml overlay gel composed of 1% agarose and 6% trypticase soy broth powder was poured on top of the previous gel, and the plates were incubated for 16 h at 37 °C. The diameters of the inhibition halos were measured to the nearest 0.1 mm and, after subtracting the diameter of the well, were expressed in inhibition units (10 units = 1 mm). We estimated the minimal inhibitory concentration (MIC) by performing a linear regression and determining the x intercepts.

Cofactor Activity of Factor H-- C3b (28 pmol) was incubated with factor I (0.16 pmol) and factor H (0.16 pmol) in the presence or absence of AM and related peptides for 24 h at 37 °C in a final volume of 50 µl of PBS. Samples were analyzed by SDS-polyacrylamide gel electrophoresis using 4-12% Tris-Bis gels (Novex) under reducing conditions and Coomassie Blue staining. C3b and factor I were purchased from Advanced Research Technologies (San Diego, CA).

Statistical Analysis-- The MTT assay values and the MIC values were analyzed by the Student's t test. cAMP values were analyzed with a one-way analysis of variance and Tukey's test. p < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Development of a Novel Nonradioactive Ligand Blotting Assay for AMBP Detection-- Using the radioligand blotting technique originally described by Hossenlopp et al. (11), we have previously demonstrated that human plasma contains at least one adrenomedullin-binding protein (AMBP-1) with Mr 120,000 under nonreducing conditions (10). In the present study, we have developed a nonradioactive ligand blotting assay. Our initial approach included the labeling of AM with three different reporters: biotin, fluorescein, or dinitrophenyl. With all of these tracers, we were able to detect AMBP-1 in human plasma (data not shown). We discarded the use of the biotinylated AM reagent due to the possible interference with avidin- or biotin-like proteins present in plasma; this problem has already been described in the development of a nonradioactive ligand blotting assay for insulin-like growth factor-binding proteins (IGFBPs) using biotinylated insulin-like growth factor I (23). The procedure using fluorescein-labeled AM gave a better signal/noise ratio than the dinitrophenyl tag and was used for the evaluation of AMBP-1 expression. Fig. 1A compares the radioligand blotting using 125I-AM or fluorescein-labeled AM. One band with Mr 120,000 was visualized in both systems; however, the nonradioactive technique resulted in sharper band definition. To demonstrate the specificity of the assay, fluorescein-labeled AM was incubated with 5 µM AM, the gene-related peptide PAMP, and the structurally related peptide CGRP (Fig. 1B). Only AM was able to displace the nonradioactive tracer. When the competition was carried out with different fragments of AM, only the intact AM molecule reduced the binding (Fig. 1C).



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Fig. 1.   Development of a new nonradioactive ligand blotting assay. A, radioligand blotting with 125I-AM is compared with a nonradioactive method using fluorescein-labeled AM (f-AM). B, competition assays with unlabeled AM, the gene-related peptide PAMP, and the structurally related peptide CGRP. C, competition assays with different fragments of AM.

Isolation and Characterization of AMBP-1-- Human plasma (2.5 ml) was fractionated by reverse phase HPLC (Fig. 2A). Each fraction was tested for the presence of AMBP-1 using the nonradioactive ligand blotting technique. AMBP-1 was revealed in fractions 48-51 (Fig. 2C). Glycoprotein staining of AMBP-1 on SDS-polyacrylamide gels revealed that AMBP-1 was glycosylated (Fig. 3A). Isoelectric focusing showed a pI 6 for the protein (Fig. 3B). For the identification of purified AMBP-1, we used three different techniques: total amino acid analysis, Edman degradation, and mass spectrometry. Table I shows the amino acid composition of AMBP-1. A data base search revealed complement factor H as the protein with the highest degree of similarity to this composition profile. The amino acid composition of factor H is also shown in Table I. The percentage of methionine was lower than expected; however, the recovery of methionine in the case of bovine serum albumin, used as a control protein, was approximately half of the expected value. The analysis of the N-terminal amino acid sequence of AMBP-1 yielded a mix of at least two main N-terminal sequences; the data base search gave us again factor H as the protein with highest homology. The sequence of the 15 amino acids analyzed was identical to the N-terminal sequence of factor H with the exception of the threonine in position 12. Furthermore, a new search with the amino acids obtained from the segment peptide that did not correspond to the N terminus of factor H showed a 65% homology with an internal sequence of factor H (residues 578-592), suggesting that this secondary sequence could correspond to factor H fragmentation. Finally, the peptide masses obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after tryptic digestion of AMBP-1 corresponded unequivocally to the tryptic digestion of factor H, with the detected peptides covering 21.8% of the total factor H sequence. MS/MS applied to the ion with a molecular mass of 1396.62 Da yielded the sequence, confirmed by both the b and the y series, of 10 out of the 12 amino acids from the fragment 737-748 in the factor H molecule.



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Fig. 2.   Isolation of AMBP-1. A, fractionation of human plasma by reverse phase HPLC. The dotted line indicates the acetonitrile gradient. Coomassie staining (B) and ligand blotting (C) of the HPLC fractions 47-51 are shown.



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Fig. 3.   Biochemical characterization of AMBP-1 as human factor H. A, Coomassie Blue and glycoprotein staining (GelCode Glycoprotein Staining Kit; Pierce) after SDS-polyacrylamide gel electrophoresis. Lane 1, horseradish peroxidase (5 µg), a glycosylated protein used as positive control. Lane 2, soybean trypsin inhibitor (5 µg), a nonglycosylated protein used as negative control. Lane 3, AMBP-1 (2 µg). B, samples were run in a pH 3-10 isoelectric focusing gel (Novex, San Diego, CA) and stained with Coomassie Blue. Lane 1, isoelectric focusing markers. Lane 2, AMBP-1 (4 µg). C, Western blot with an anti-factor H antibody. Lane 1, AMBP-1 (100 ng). Lane 2, AMBP-1 (200 ng). Lane 3, commercially available human factor H (50 ng). Lane 4, human plasma (0.2 µl). D, nonradioactive ligand blotting of 1 µg of AMBP-1 (lane 1) and factor H (lane 2). E, ligand blotting of AMBP-1 (250 ng) run under unreduced (lane 1) or reduced conditions (lane 2). F, binding of fluorescein-labeled AM (50 nM) to a 96-well plate coated with factor H (5 ng/well) was competed with increasing concentrations of unlabeled AM (). Neither PAMP (*) nor CGRP (open circle ) affected the binding. The results of one of two independent experiments are shown. Values represent mean and S.D. of three determinations. B/B0 represents the ratio of signals generated in the presence/absence of unlabeled peptide.


                              
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Table I
Amino acid composition of AMBP-1 and factor H
The amino acid composition of factor H was obtained from the protein sequence database SWISS-PROT (accession number: P08603).

To further confirm that AMBP-1 was in fact factor H, we analyzed the ability of anti-factor H antibody to recognize AMBP-1 by Western blot detection. AMBP-1 was immunoreactive for this antibody, giving a band of Mr 120,000 under nonreducing conditions, an identical size to that of commercially available human factor H (Fig. 3C). Moreover, with nonradioactive ligand blotting using fluorescein-labeled AM, we demonstrated that factor H binds to AM (Fig. 3D). Under reducing conditions, AMBP-1 had a Mr of 150,000 (Fig. 3A), which corresponds to the Mr reported for factor H in such conditions (24). The reduction of the disulfur bonds prevented the binding of AMBP-1 to the fluorescein-labeled AM (Fig. 3E).

Finally, we carried out binding assays of fluorescein-labeled AM to immobilized factor H. Fig. 3F shows the competitive displacement with unlabeled AM that could not be achieved with either CGRP or PAMP.

Interference of Factor H with the AM Radioimmunoassay-- We previously reported that the C18 reverse-phase separation technique used to prepare plasma for AM RIA analysis effectively eliminates AMBP-1 from the extract (10). In the present work, we confirmed this observation by analyzing the presence of factor H in plasma before and after the C18 extraction. When plasma is processed through the C18 column, factor H is obtained in the unbound portion of the sample and not in the fraction used for AM quantification (Fig. 4A). Based on this observation, we tested the possibility that a certain amount of AM may pass through the column bound to factor H. For that purpose, 1 ml of human plasma was processed through the column, and both the bound and unbound extracts were recovered. We immunoprecipitated AM from the extracts and determined its presence by Western blotting. AM was detected in both the unbound and the bound fractions (Fig. 4B), suggesting that the traditional procedure used for peptide purification does not recover the total amount of AM present in plasma. Western blot after immunoprecipitation in the absence of extract did not yield any band, excluding the possibility that AM comes from the rabbit anti-AM serum (data not shown).



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Fig. 4.   Western blot for factor H and AM after C18 extraction. A, 1 ml of human plasma was processed through a Sep Pak C18 cartridge, and both the bound and unbound fractions were analyzed for factor H presence by Western blot. Lane 1, commercially available human factor H (10 ng). Lane 2, whole human serum (0.5 µl). Lane 3, unbound fraction (1 µl). Lane 4, bound fraction (1 µl). B, the same was done for the detection of AM (immunoprecipitating AM before the Western blot analysis). Lane 1, synthetic AM (1 ng). Lane 2, fraction immunoprecipitated with normal rabbit serum (30 µl). Lane 3, fraction immunoprecipitated with rabbit anti-AM antibody (30 µl).

Disruption of AM/factor H interaction demonstrated a second source of AM in the plasma that was not routinely accounted for by traditional C18 purification procedures prior to RIA determination. A ligand blotting was performed with purified AMBP-1. After incubation with fluorescein-labeled AM, we tested several conditions for the dissociation of the binding between AM and factor H. For that purpose, the membrane was cut in strips and washed six times for 10 min each under the different conditions. Finally, the strips were equilibrated again in the assay buffer, and the ligand blotting was developed (Fig. 5A). Extreme conditions such as acidic pH and high salt concentration did not dissociate the interaction of factor H with AM. However, the incubation of the blot with labeled AM in those conditions effectively avoided the interaction (data not shown). One of the treatments that disrupted the binding was basic pH; however, further experiments indicated that this effect could in fact correspond to an artifact, since this extreme pH alters the structure of the fluorescein and doing so negates its affinity for the anti-fluorescein antibody (data not shown). Using the binding assay in a 96-well plate, we further analyzed the dissociating ability of the chaotropic agent sodium thiocyanate (NaSCN). After incubation of solid-phased factor H (50 ng) with fluorescein-labeled AM (50 nM) and prior to the development of the assay, wells were incubated during different periods of time with PBS, 3 M NaSCN, pH 7.4 (Fig. 5B). The displacement curve suggests two dissociation events. By 15 min, the chaotropic salt had already displaced 50% of the binding; however, after that point the dissociation rate decreased.



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Fig. 5.   Dissociation of factor H/AM interaction. A, purified AMBP-1 (fraction 48) was electrophoretically fractionated and transferred to a membrane. After incubation with fluorescein-labeled AM and prior to the final development, the membrane was incubated under different conditions (neutral pH unless indicated). Lane 1, PBS; lane 2, pH 11.5; lane 3, pH 2.5; lane 4, 4 M NaCl; lane 5, 4 M NaCl, pH 11.5; lane 6, 4 M NaCl, pH 2.5; lane 7, 1% SDS; lane 8, 3 M urea; lane 9, 3 M guanidine-HCl; lane 10, 3 M sodium thiocyanate; lane 11, 50% ethylenglicol, pH 11.5; lane 12, 50% ethylenglicol; lane 13, 1% beta -mercaptoethanol. B, binding assay in a 96-well plate. After incubation with fluorescein-labeled AM and prior to the development of the assay, wells were incubated during different periods of time with PBS, 3 M NaSCN, pH 7.4. Values represent the mean and S.D. of six determinations. B/B0 represents the percentage of total binding.

Plasma samples of three healthy donors were quantified by RIA following either the method previously described (18, 19) or the following modification. 1 ml of plasma was preincubated with an equal volume of 6 M NaSCN in PBS, 0.1% ATC, 0.1% Triton X-100, pH 7.4, for 10 min at room temperature. After that incubation, plasma was extracted through the C18 cartridges and quantified. The detected levels with the new protocol were 2-fold higher than those obtained with the standard technique (mean and S.D. values of the three donors were 23.0 ± 4.8 versus 54.3 ± 8.6 pg/ml). The same results were obtained when a longer preincubation with the chaotropic agent (16 h at 4 °C) was used. Using the new protocol with NaSCN, recovery of unlabeled AM (200 pg) added to human plasma was 93.9 ± 18.7% (n = 3), whereas recovery of 125I-AM was 82.7% ± 4.4% (n = 6). The dilution curve in the RIA obtained from a fourth plasma sample was parallel to that of the synthetic human AM used in the standard curve and to the curve generated with the same plasma extracted under the traditional conditions (Fig. 6). The parallel curves confirm that the increase in AM immunoreactivity generated by NaSCN treatment was not artifactual.



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Fig. 6.   Competitive binding curves generated by human plasma in the AM radioimmunoassay. The dilution curves of plasma (4 ml) extracted following the standard protocol (+) or the NaSCN modification () were compared with the standard curve of synthetic AM (open circle ). B/B0 represents the ratio of radioactivity bound to that bound in the absence of added standard. The scale bar over the curves represents the different plasma dilutions.

Effect of Factor H in AM-mediated cAMP Induction-- AM was initially identified as a peptide capable of elevating cAMP (1). Recently, it has been reported that Rat-2 fibroblast cells express a specific AM receptor coupled to adenylate cyclase that produces a dose-dependent increase in cAMP upon exposure to AM (20). Using this cell line as a model, we studied the effect that factor H could have on AM-mediated cAMP response. Treatment of Rat-2 fibroblasts with 100 nM AM produced a 2-fold increase in cAMP (Fig. 7A). When the same concentration of AM was combined with increasing concentrations of factor H (50, 100 and 200 nM), we observed a significant and dose-dependent augmentation in cAMP production. The highest factor H concentration (200 nM) used alone had no effect on cAMP levels, confirming that the observed increase was in all cases due to the presence of AM (Fig. 7A). On the other hand, the presence of factor H did not apparently modify the kinetics of the binding between rat AM and its receptor. Neither the association rate nor the competitive binding of human AM was altered by the presence of factor H (Fig. 7, B and C).



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Fig. 7.   Effect of factor H in AM activity on Rat-2 cells. A, effect of factor H in AM-mediated cAMP induction. cAMP was measured after incubating Rat-2 cells with AM and increasing concentrations of factor H. Incubations with AM and 100 or 200 nM factor H produced a significant increase in cAMP as compared with the levels obtained with AM alone (p < 0.01 (**) and p < 0.001 (***), respectively). cAMP levels after incubation with factor H alone did not significantly differ from the basal conditions. Values represent mean and S.D. of four independent determinations. B, association rates of AM to Rat-2 cells. Confluent Rat-2 cells were incubated with rat 125I-AM (100 pM) in the absence () or presence of 10 nM human factor H (open circle ). At the indicated times, bound 125I-AM was determined. Specific binding was obtained by subtracting the nonspecific binding from the total binding. Nonspecific binding was measured by incubating the cells with a 1000-fold excess of unlabeled AM. Values represent mean and S.D. of three independent determinations. C, competition curves for AM binding in Rat-2 cells. Rat 125I-AM (100 pM) was incubated with Rat-2 cells and increasing concentrations of human AM in the absence () or presence of 10 nM human factor H (open circle ). Specific binding was calculated as described above. Values represent mean and S.D. of three independent determinations.

Effect of Factor H in AM-induced T-47D Growth-- A human breast cancer cell line, T-47D, was used to investigate the effect of AM and factor H interaction on tumor cell growth. We have previously demonstrated that AM can act as an autocrine/paracrine growth factor in several cancer cell lines (25). In the present work, we used the MTT proliferation assay to examine the growth-promoting activity of AM in the presence or absence of factor H. In a serum-free medium, AM had a growth-promoting activity on the cell line T-47D (data not shown). The presence of factor H further induced the proliferation of T-47D in a dose-dependent manner. Factor H in the absence of AM had no effect on growth (Fig. 8).



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Fig. 8.   Effect of factor H in the AM-induced proliferation of the breast cancer cell line T-47D. The cells were incubated in serum-free medium with increasing concentrations of human factor H in the absence () or presence of 2 µM human AM (open circle ). Values are expressed as percentage of growth induction as compared with the growth in the absence of factor H. All factor H treatments showed a significant induction of growth (p < 0.001). The mean and S.D. of eight values are represented.

Modulation by Factor H of the Antimicrobial Activity of AM-- A radial diffusion assay was used to characterize the influence of human complement factor H on the antimicrobial activity of AM (Fig. 9). Factor H by itself did not have any antimicrobial effect on E. coli. On the other hand, AM had an intense inhibitory impact on the bacterial growth. When AM and factor H were added together, a significant reduction in the inhibitory effect of AM was observed (p < 0.001, n = 8), suggesting that factor H is able to hinder the antimicrobial activity of AM. The MIC for AM by itself was 18.4 ± 1.3 µg/ml, and it became 35.4 ± 1.1 µg/ml when factor H (50 µg/ml) was added.



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Fig. 9.   Antimicrobial effect of AM (), factor H (*), or AM in the presence of 50 µg/ml of factor H (open circle ). The results are expressed in inhibition units (10 units correspond to 1 mm of diameter in the inhibition halo). MIC values were estimated by performing a linear regression and determining the x intercepts. The MIC value for AM was 18.4 ± 1.3 µg/ml, and it became 35.4 ± 1.1 µg/ml when factor H was added (p < 0.001). The values represent mean and S.D. of the mean of eight determinations.

Modulation by AM of the Cofactor Activity of Factor H-- We finally tested whether AM affects the cofactor activity of factor H in the factor I-mediated cleavage of C3b (26, 27). Treatment of C3b with factor H and factor I caused cleavage of C3b (Fig. 10). When AM was added to the reaction, an increase in the cleavage of C3b was observed (Fig. 10A). Note that as the levels of AM are increased in the reaction mixture, there is a parallel increase in the split product formation with a reciprocal reduction in the 104-kDa band. AM (10 µM) had no activity in the absence of factor H. Neither CGRP nor PAMP (10 µM) had any effect on the cofactor activity (Fig. 10B).



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Fig. 10.   Effect of AM in the cofactor activity of factor H. C3b (104-kDa alpha ' chain and 71-kDa beta  chain) was incubated 24 h at 37 °C with factor H, factor I, and different peptides. The cleavage of the C3b alpha ' chain produced three bands with Mr 68,000, 43,000, and 42,000. A, effect of different AM concentrations in the cofactor activity of factor H. B, effect of AM compared with the effect of the structure related peptide CGRP and the gene-related peptide PAMP. Each panel shows a representative example of three different experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We demonstrate here that AMBP-1 circulating in human plasma corresponds to complement factor H. The purification of AMBP-1 from plasma was greatly facilitated by a novel nonradioactive assay for the detection of AM binding proteins. This new technique gave us some distinct advantages over our previously described radioactive method (10); its development required a shorter period of time, and the use of the fluorescein-labeled AM had better reproducibility and sharper band formation than the use of 125I-AM. In addition, it simplified handling procedures and extended the half-life of the tracer. Since both methods revealed a protein with the same molecular weight and the binding with the labeled AM could only be totally displaced by the intact unlabeled AM, we conclude that the protein detected with the nonradioactive method corresponds to the protein previously detected with the radioactive ligand (10).

By a combination of  HPLC, electrophoretic fractionation, and the nonradioactive detection system, we have been able to isolate AMBP-1 to homogeneity and complete its biochemical identification. Several different analytical techniques led to the unequivocal conclusion that the purified protein corresponds to complement factor H. Factor H is a single chain glycoprotein consisting of 20 subunits called short consensus repeats (28). Factor H binds to C3b, displacing Bb from the C3 convertase. It also acts as a cofactor for the factor I-mediated proteolytic cleavage of the alpha ' chain of C3b. The final result of these activities is the inhibition of the alternative pathway of the complement (24, 26, 27). Additional roles have been identified for factor H; it binds to the integrin Mac-1 (C11b/CD18) enhancing the activation response of human neutrophils (29), is a ligand for L-selectin (30), induces the secretion of interleukin 1beta by human monocytes (31), and acts as a chemotactic protein for monocytes (32). Finally, factor H binds to cell surface components of several pathogens (33-37). This binding apparently inhibits the activation of the complement, thus enhancing the pathogenicity of these microorganisms.

The discovery of the interaction between AM and factor H raises many questions about its biological implications. The presence of a binding protein can limit the transport of a peptide to the interstitial space and the access to its specific receptors; it can protect a peptide from metabolic clearance events, thereby prolonging its half-life in circulation; and it can modulate its biological activity. Here we show some of the consequences of the binding between AM and factor H.

One of the most immediate aspects was the interference of factor H with the routine AM quantification assay. The procedure for AM determination requires an extraction step to avoid interferences in the RIA. This process eliminates factor H from the extract to be analyzed, suggesting that factor H may be responsible for the interferences in the nonextracted samples. Although factor H is more hydrophobic than AM and therefore is better retained by the reverse-phase matrix,2 we have demonstrated that in the established protocol for AM extraction, factor H is not retained by the Sep-Pak C18 cartridges. This fact could be related to the physical characteristics of the cartridges. Factor H is a molecule with a contour length of 495 Å and a cross-sectional diameter of 34 Å that folds on itself, reducing the length of the protein and increasing its width (38). Factor H is not retained in the C18 matrix, probably because it is too big to penetrate through the particle pores (the pore size is 125 Å based on the manufacturer's specifications). The way factor H circulates through the column suggests that the AM bound to factor H will not be retained and therefore the extraction protocol would recover only the free AM in plasma. We have confirmed this by demonstrating the presence of a significant amount of AM in the unbound fraction after the extraction. Furthermore, treatment with a chaotropic agent (NaSCN) seems to dissociate, at least partially, the binding between factor H and AM, allowing the detection of higher levels of AM. Plasma levels of AM are elevated in several pathological conditions (39), and although AM seems to act in an autocrine/paracrine manner, a physiological role for circulating AM remains possible (40). Therefore, we believe that determining the total AM concentration in plasma (versus the free AM currently measured) may be important to better understand the role of AM in the physiological and pathological conditions in which it is implicated. In addition, the variations in the levels of AMBP previously observed in infected animals (10) suggest that changes in circulating AM may be also dependent on modifications of its binding protein expression.

Factor H is present in plasma and has also been detected in extravascular compartments such as the synovial fluid (41, 42). The liver is considered to be the main source of factor H, although it is also synthesized by extrahepatic cells such as mononuclear phagocytes, fibroblasts, endothelial cells, mesangial cells, astrocytes, oligodendrocytes, and neurons (43). This suggests that the presence of factor H in tissues may affect the autocrine/paracrine actions of AM. We describe here preliminary insights into the effect of factor H on AM activity. An increase in the cAMP induction mediated by AM was observed when Rat-2 fibroblasts were incubated with AM in the presence of factor H. On the other hand, factor H did not affect the binding between AM and its receptor. Factor H was also able to augment the growth-promoting activity of AM on the human breast cancer cell line T-47D. The exact mechanism by which the factor H-AM complex augments AM activity remains to be clarified; however some observations may shed some light on this issue. Factor H is able to bind to cell surfaces through at least three glycosaminoglycan binding sites present in its structure (44-46). It has also been reported that factor H binds to human neutrophils through the integrin Mac-1 (C11b/CD18) (29), and it is a ligand for L-selectin (30). Hypothetically, the augmentation in cAMP production and cell growth may involve enhanced cell surface attachment, presenting AM in closer proximity to its membrane receptor. Factor H would act as a carrier and a reservoir of AM, which could provide high local levels of AM to stimulate its receptor. In this way, factor H would increase the AM effectiveness without modifying the affinity for its receptor. Other binding proteins enhance the biological activity of their ligands; the case of the well characterized IGFBPs is a good example. IGFBPs can either inhibit or augment the IGF actions (47). How IGFBPs enhance IGF function is not well understood, although it is known that many IGFBPs associate with cell surfaces and that the enhancing activity is probably mediated by this binding (47). A similar example of this phenomenon can be seen with the latent transforming growth factor-beta (TGF-beta )-binding protein (LTBP), which seems to play an important role in the activation of latent TGF-beta , probably through targeting the latent TGF-beta complex to the cell surface (48). The presence of an Arg-Gly-Asp (RGD) sequence may account for the cell attachment properties of some IGFBPs (47) as well as the LTBP (49). This sequence is present in several matrix proteins, and it is the essential structure recognized by the integrin superfamily of receptors (50). Interestingly, factor H also possesses an RGD cell adhesion sequence in its structure (28). Finally, it would also be interesting to determine whether the enhancing effect of factor H on Rat-2 cAMP production and on T-47D growth may be due to a protective effect of factor H on AM degradation.

We have also been able to demonstrate that factor H down-regulates the antimicrobial activity of AM. It has been postulated that AM exerts its bactericidal effect by forming membrane pores, which ultimately cause pathogen lysis (51). Factor H inhibition of AM antimicrobial activity could be mediated by decreasing the concentration of available AM in the microenvironment, thus limiting its access to the pathogen's outer membrane. Although the physiological relevance of AM's antimicrobial activity still has to be addressed, the inhibition by factor H of this AM activity is in line with the fact that the binding of factor H by certain microorganisms seems to protect them from complement-mediated host defense (33). It has been suggested that this resistance could be due to the degradation of C3b by the membrane-bound factor H. The inactivation by factor H of the host-produced AM, a molecule with antimicrobial activity, could now be considered as an additional mechanism of microorganism resistance.

Another important aspect of this study is the identification of a role for AM in C3b degradation mediated by factor H/factor I interaction. We have demonstrated that AM accelerates this process. We can speculate that AM may induce conformational changes in the structure of factor H, increasing its affinity for C3b, similarly to what has been reported to occur in the presence of soluble polyanions (52). Since plasma AM is increased during sepsis and after endotoxin challenge (12, 53-56), AM induction of factor H/factor I-mediated C3b degradation may have important implications in the immune response associated with these processes.

Although we have addressed some of the consequences of the factor H/AM interaction, many important questions remain. Do factor H and AM affect the production of their binding partner? How does factor H affect the biological actions of AM in vivo? Does factor H prolong the plasma half-life of AM? In regard to this last question, it is interesting to note that the recovery of endogenous AM from plasma is not affected by several freeze-thaw cycles; however, recovery of exogenous AM is markedly reduced after a single cycle (18). The authors of this work suggested that a protein may be present in circulation that could bind AM aiding in its stability in plasma. It would not be surprising if that protein was factor H.

In summary, we present here the interaction between factor H and AM and some of the resulting consequences it may have on the activities of both protein partners. We have shown the influence that factor H/AM binding has on established AM quantitative techniques traditionally used in the field. These observations challenge our understanding of the real significance of free AM in biological fluids. Factor H has been shown to affect AM biology in a diametric manner, depending on the experimental system examined. The exact mechanisms by which these actions occur remain to be determined. In addition, AM can modulate factor H activity during degradation of C3b, thereby implicating this peptide hormone as a new regulatory component of the complement cascade. Thus, our initial observation on the existence of an AM plasma binding protein has now opened diverse avenues for future studies in both AM and factor H biology.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (Spain) Grant 98/9172. To whom correspondence should be addressed: Dept. of Cell and Cancer Biology, National Cancer Institute, NIH, Bldg. 10 Rm. 13N262, Bethesda, MD 20892. Tel.: 301-402-3308; Fax: 301-435-8036; E-mail: pior@mail.nih.gov.

Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M007822200

2 R. Pío, A. Martínez, E. J. Unsworth, J. A. Kowalak, J. A. Bengoechea, P. F. Zipfel, T. H. Elsasser, and F. Cuttitta, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: AM, adrenomedullin; CGRP, calcitonin gene-related peptide; PAMP, proadrenomedullin N-terminal 20 peptide; AMBP, adrenomedullin binding protein; AMBP-1, human AMBP; RIA, radioimmunoassay; ATC, alkali-treated casein; TBS, Tris-buffered saline; HPLC, high pressure liquid chromatography; MIC, minimal inhibitory concentration; IGFBP, insulin-like growth factor-binding protein; TGF-beta , transforming growth factor-beta ; LTBP, TGF-beta -binding protein; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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