From the 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
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
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ABSTRACT |
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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.
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.
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%
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 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.
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).
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.
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).
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.
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.
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).
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).
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.
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).
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 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- 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (76K):
[in a new window]
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.
View larger version (43K):
[in a new window]
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 (
)
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.
Amino acid composition of AMBP-1 and factor H
View larger version (41K):
[in a new window]
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).
View larger version (32K):
[in a new window]
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%
-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.
View larger version (16K):
[in a new window]
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 (
). 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.
View larger version (23K):
[in a new window]
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 (
). 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
(
). Specific binding was calculated as described above. Values
represent mean and S.D. of three independent determinations.
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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 (
). 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.
View larger version (16K):
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Fig. 9.
Antimicrobial effect of AM ( ), factor H
(*), or AM in the presence of 50 µg/ml of
factor H (
). 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.
View larger version (37K):
[in a new window]
Fig. 10.
Effect of AM in the cofactor activity of
factor H. C3b (104-kDa ' chain and 71-kDa
chain) was
incubated 24 h at 37 °C with factor H, factor I, and different
peptides. The cleavage of the C3b
' 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
' 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 1
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.
(TGF-
)-binding protein (LTBP), which seems to play an important role in the activation of latent TGF-
, probably through targeting the latent TGF-
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.
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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.
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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-, transforming growth factor-
;
LTBP, TGF-
-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.
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