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INTRODUCTION |
In response to tissue injury or infection, activated macrophages
secrete cytokines (interleukin-1 and -6 and tumor necrosis factor) that
induce liver synthesis of a number of acute-phase (AP)1 proteins (1, 2). The
function of most of the AP proteins is unknown, but it is widely
accepted that their purpose is to enhance host survival by neutralizing
infectious agents, contributing to tissue repair, and restoring
homeostasis. One of these AP proteins is a novel HDL apolipoprotein,
called serum amyloid A (apoSAA), which is encoded by a multigene family
conserved from fish to humans (3-5). Maximum transcription rates are
reached for two of the four known apoSAA isoforms (apoSAA1 and apoSAA2)
3 h after AP induction (6-8). Their concentration in plasma
increases 500-1000-fold (from 1-5 µg/ml up to 1 mg/ml) within
18-24 h and returns to near normal levels within 5-7 days of a single
inflammatory stimulus (9). ApoSAA is found mainly associated with
HDL3 (10, 11), where it has been postulated to modulate
cholesterol transport during the AP response (12, 13). But several
other functions have also been proposed for apoSAA including immune
regulation, as a chemoattractant and as an inhibitor of fever
induction, platelet activation, and neutrophil oxidative burst (3).
ApoSAA was originally identified by its cross-reactivity with antisera
raised against peptides isolated from inflammation-associated amyloid
(AA amyloid) (14, 15) which is a pathological tissue deposit associated
with chronic inflammatory diseases. Amyloid is a generic term
describing the primarily extracellular accumulation of fibrillar
protein deposits that have unique tinctorial and structural properties
and that cause the disruption of tissue architecture and function (16,
17). ApoSAA and at least 17 other unrelated normally nonfibrillar
proteins are known precursors of amyloid (18). Each is associated with
a specific disease such as Alzheimer's disease, chronic hemodialysis,
adult-onset diabetes, rheumatoid arthritis, and certain malignancies.
Regardless of the underlying amyloid fibril protein/peptide or
associated disease, isolated amyloids fibrils are composed of two or
more 3-nm filaments twisted around each other forming nonbranching fibrils, 7-10 nm in diameter, with a crossed
-pleated sheet
conformation. They stain with Congo Red, and when stained and viewed
under polarized light they exhibit a red/green birefringence, a
property diagnostic for amyloid.
It has been proposed that the deposition of amyloid requires the
formation of a nidus or protofilament around which amyloid fibrillogenesis takes place, and the glycosaminoglycan (GAG) heparan sulfate (HS) plays an important role in this pathological process (17,
19, 20). GAGs are sulfated heteropolysaccharides that have been known
to be associated with amyloid for over 30 years (21, 22) but attracted
little attention until 1987, when Snow and co-workers (23) showed that
the GAG component of mouse AA amyloid was deposited coincidentally with
the amyloid protein and that the GAG was part of the HS proteoglycan,
perlecan (24, 25). By experimentally varying the induction speed of
murine AA-type amyloidosis, it was possible to show that HS was both temporally and spatially deposited with the AA peptide (24-26) and
that splenic perlecan mRNA was increased prior to the histological detection of AA amyloid (26). In vitro experiments have also shown that of a number of different GAGs examined, only HS could increase the
-sheet content (the characteristic conformation of
amyloid) for mouse apoSAA2 but not for the nonamyloidogenic isoforms
apoSAA1 and apoSAACE/J (27, 28).
HS has in fact been found in all amyloid deposits that have been
investigated (19). High affinity binding between perlecan and three
Alzheimer's amyloid (A
) precursors,
PP-695,
PP-751, and
PP-770, could be inhibited with dextran sulfate and heparin but not
chondroitin sulfate or dermatan sulfate (29). HS has also been found to
enhance A
fibrillogenesis (30), and analogs of HS
(aliphatic polysulfonates) were recently reported to block HS-induced
A
fibrillogenesis, in vitro, and interfere with in vivo AA-type amyloid accumulation in mice (31, 32). Congo Red
(CR), a disulfonated acidic dye, which has long been used as a stain
for amyloid (33), can inhibit AA amyloid in vivo (34). CR
and a number of sulfated glycans have also been shown to prevent the
accumulation of the protease-resistant prion protein (amyloid form)
(35-37).
HS and closely related heparin are negatively charged polymers composed
of disaccharide repeats that contain carboxyl and sulfate groups (38,
39). Many proteins bind these GAGs through electrostatic interactions,
and it has been demonstrated by substitution and chemical modification
experiments that the protein binding is dependent on their positively
charged (basic) residues (40-42). Because a wide range of proteins
bind specifically to heparin, it was expected that a common structural
motif would be found. Cardin and Weintraub in 1989 (43) examined a
series of heparin-binding sequences and found that the basic residues
tended to be arranged on one side of an
-helix with the pattern
XBBBXXBX (B, basic residue; X,
nonbasic residues). A second pattern,
XBBXBX, was proposed to align the
basic residues on one side of a
-strand. Later, a third consensus
sequence was published, XBBXXBBBXXBBX (44). However, there are many examples of heparin/HS-binding sequences
lacking these consensus sequences, and recently Margalit et
al. (45) reported that a common feature of heparin-binding sequences was that the outer basic residues were always 20 Å apart.
The co-localization of HS with amyloids, the induction of perlecan
expression prior to the appearance of AA amyloid, its ability to
promote conformational change in native amyloid precursors and amyloid
fibrillogenesis in vitro, and the amyloid-blocking effects
of HS analogs and other sulfonates are all consistent with the working
hypothesis that HS-amyloid precursor associations take place in
situ and are a critical early step leading to protofilament formation and/or amyloid fibrillogenesis. In an effort to understand the underlying mechanism of HS-dependent conversion of
apoSAA into AA amyloid fibrils, we undertook this study to characterize the GAG binding activity of apoSAA. This was done by affinity chromatography with defined apoSAA peptides using columns to which different GAGs were covalently attached. The GAG-binding site on apoSAA
was initially mapped testing the binding activities of apoSAA CNBr
fragments. This was followed with an assessment of the binding
activities of a series of synthetic peptides corresponding to the
smallest heparin/HS-binding CNBr fragment, containing specific residue
substitutions, or a deletion. This approach allowed us not only to
identify the heparin/HS-binding peptide sequence but also to rank the
relative importance of the individual basic residues within the binding
site. The demonstration of heparin/HS binding activity for apoSAA is
consistent with some of the functions proposed for apoSAA.
Characterization of the apoSAA-binding site also advances our
understanding of amyloidogenesis and may assist in the design of
therapeutic compounds.
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EXPERIMENTAL PROCEDURES |
Purification of Lipoproteins and ApoSAA Peptides--
Plasma
apoSAA concentrations were experimentally elevated in CD1 mice (Charles
Rivers, Montreal, Quebec, Canada) by a subcutaneous injection of 0.5 ml
of 2% (w/v) AgNO3 (46) which resulted in a sterile
abscess. After 18-20 h, mice were sacrificed by CO2 narcosis and exsanguinated by cardiac puncture preventing clotting with
a small amount (50 µl) of 7% EDTA. High density lipoprotein containing apoSAA (HDL-apoSAA) was isolated from plasma by density flotation (47). The density of the plasma was adjusted to 1.25 g/ml
with NaBr and centrifuged at 250,000 × g for 24 h
at 10 °C. The top layer was aspirated, pooled, and LDL and
HDL-apoSAA were separated by gel filtration on a Sephacryl S-300 column
(1.5 × 45 cm) washed with 50 mM Tris, 150 mM NaCl, pH 7.5, at 20 ml/h. Normal HDL and LDL were also
purified from uninflamed mice by this procedure.
ApoA-I and apoSAAs were purified from total lipoprotein by gel
filtration as described previously (48). The lipoprotein preparations
(3-5 ml) were dialyzed against 10% formic acid, pH 2.0 (2 liters),
for 18 h at 4 °C and then applied to a Sephacryl-S-100HR column
(2.5 × 110 cm) and eluted in the same buffer at 25 ml/h. The
sample separated into two peaks which were collected, frozen in liquid
N2, lyophilized, and stored at
20 °C. The protein
residue from the first peak containing apoA-I was delipidated with
diethyl ether (49). The dried residue was resuspended in 4 M urea, 10% formic acid, and the apoA-I was purified by
gel filtration on the Sephacryl S-100HR column.
Individual apoSAA isoforms were purified from the second gel filtration
peak by reversed phase-high performance liquid chromatography (RP-HPLC)
using a Waters (Millipore) HPLC system with a model 680 automated
gradient controller, model 501 pump units, and a series 440 absorbance
detector connected to a Waters 740 data module integrator. The
lyophilized apoSAA powder was solubilized in 20% formic acid and then
injected onto a semi-preparative C18 Vydac column (1 × 25 cm),
eluted at 3 ml/min with 0.1% trifluoroacetic acid, 10% acetonitrile
for 5 min, and then developed with an acetonitrile concentration
gradient, increasing 2.5%/min for 10 min followed by 1.0%/min for 20 min, and finally 4.3%/min for 10 min bringing the elution buffer to
98% acetonitrile by 45 min. The eluant was monitored continuously at
214 nm, and absorbance was plotted against retention time. Peaks were
collected manually and dried by vacuum centrifugation. The dried
residue was stored at
20 °C. Apolipoproteins were identified by
their mobility on SDS-urea-polyacrylamide gel electrophoresis (50).
Preparation of the ApoSAA Peptides--
ApoSAA1 and apoSAA2 were
cleaved at Met-X peptide bonds with cyanogen bromide (CNBr).
Protein was dissolved in 70% formic acid at 1 mg/ml, to which 5.5 mg/ml CNBr was added (250 M excess over Met) plus free
L-Trp (5 M excess over Met) to protect Trp residues. The reaction was carried out under nitrogen, at room temperature overnight, and then the solvent was evaporated by vacuum
centrifugation. The reaction was evaluated, and peptides were purified
by RP-HPLC. A semi-preparative C-18 Vydac column was equilibrated with
10% acetonitrile, 0.1% trifluoroacetic acid. Peptides were dissolved
in 40% formic acid, filtered through a 0.2-µm filter, or centrifuged
at 10,000 × g, and loaded onto the column, washed for
5 min at 3 ml/min, and then developed with a 1.5%/min acetonitrile
linear concentration gradient for 30 min, followed by 4.3%/min
acetonitrile for 10 min, bringing the elution buffer to 98%
acetonitrile by 45 min. The separated peptides were identified by
amino-terminal sequencing and molecular weight determination by mass
spectroscopy carried out at the Alberta Peptide Institute (Edmonton,
Alberta, Canada). Also, a series of apoSAA peptides corresponding to 1)
mouse apoSAA2, residues 77-103 (m27-mer), with specific basic residues
substituted with Ala, 2) mouse apoSAA2, residues 77-96 (a 20-mer
missing Lys-102), and 3) human apoSAA2, residues 78-104 (h27-mer),
were synthesized by Multiple Peptide Systems (San Diego, CA).
Glycosaminoglycan, Congo Red, and Taurine Affinity
Columns--
Heparin/Affi-Gel media were purchased from Bio-Rad.
Heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and
hyaluronan were all purchased from Sigma and coupled to either to
Sepharose 4B (Amersham Pharmacia Biotech) based on the method of Smith
et al. (51) or to Affi-Gel 102 (Bio-Rad) as per
manufacturer's instructions. Sepharose 4B was washed with 20 bed
volumes of water, resuspended in 1 volume of water, and transferred to
a beaker with a stir bar on ice. GAGs were dissolved in water at 2 mg/ml and were also placed on ice. The two solutions were mixed, and while stirring the pH was adjusted to pH 11 with NaOH (5 N). Cyanogen bromide, 1 g/ml in
N,N-dimethylformamide, was added dropwise to a
final concentration of 31.3 mg/ml. The pH was maintained at about 11 by
adding NaOH for 15 min and then left stirring for 18 h at room
temperature. The gel was then washed with 20 bed volumes of water
followed by 1 M ethanolamine, pH 9.0, to block excess
reactive groups. After further washing with 10 bed volumes of (i)
water, (ii) 0.1 M sodium acetate, pH 4.0, and (iii) 0.1 M NaHCO3, pH 8.3, the column gel was
equilibrated in 20 mM Tris-HCl, pH 7.5. Affi-Gel 102 (4 ml)
was washed with 20 bed volumes of 50 mM acetate, pH 6.0, and then 8 mg of GAG in 4 ml of the same buffer was mixed with the gel.
The coupling reaction was initiated by the addition of 32 mg of
1-ethyl-3-3-dimethylaminopropyl carbodiimide, adjusting the pH to 5 with 1 N HCl and allowing the reaction to proceed for
3 h. Heparin and HS-Sepharose 4B were also treated with
carbodiimide that forms a stable adduct with GAG carboxyl side groups
(52). The amount of GAG linked to columns ranged from 0.5 to 0.75 mg/ml
gel as determined colorimetrically by the toluidine blue method (51).
Taurine (2-aminoethanesulfonate) and Congo Red (CR) dye were also
coupled to Sepharose 4B. Taurine at 30 mg/ml and CR at 5 mg/ml were
reacted with the CNBr-activated matrix. Once the reaction was complete
the CR-Sepharose 4B was washed with 10% ethanol followed by 2 M guanidine HCl, pH 7.5, to remove excess CR prior to
equilibration with 20 mM Tris-HCl, pH 7.5. Based on the
amount of CR that was washed from the column (absorbance at 480 nm),
3.8 mg of CR was coupled to the column. By including
[3H]taurine (15 µCi) with the cold taurine, the amount
of taurine coupled to Sepharose 4B was estimated at 12.4 mg/ml by
scintillography. The CR and taurine columns contained approximately
equimolar amounts of sulfate groups.
Affinity Chromatography on GAG-charged Columns--
ApoSAA CNBr
cleavage products were resuspended in 20 mM Tris-HCl, pH
7.5, and loaded onto a 6-ml heparin-Affi-Gel column equilibrated with
the same buffer at 0.5 ml/min. The column was then washed with 4 bed
volumes of buffer and then developed with a 0-1 M NaCl concentration gradient. Fractions (0.6 ml) were collected and their
absorbance measured at 214 nm. Heparin/Affi-Gel and other GAG-charged
matrices were also packed into a 3-ml stainless steel column and
equilibrated with 20 mM Tris-HCl, pH 7.5, at 0.5 ml/min using a Waters HPLC system. Samples (20-80 µg in 150-200 µl) were injected onto the column, washed with 3 bed volumes (18 min) of the
same buffer, and then developed with a 0-1.0 M NaCl linear gradient for 10 bed volumes (60 min). The eluate was monitored continuously at 214 nm, and the absorbance was plotted against the
retention time (RT). Generally, unbound peptides/proteins eluted
6.5-7.0 min after loading, and based on the RTs for the bound
peptides/proteins, the NaCl concentration at which desorption took
place could be calculated as follows: desorption [NaCl] = (RT
6.5 min
18 min)/60 min.
 |
RESULTS |
Cyanogen Bromide Cleavage of RP-HPLC-purified ApoSAA1 and
ApoSAA2--
The acute-phase serum amyloid A isoforms, apoSAA1 and
apoSAA2, of mouse are 91% identical in amino acid sequence and have a
single heparin-binding consensus sequence
(XBBXBX, X, non-basic residue; B, basic residue) (43) located between residues 82 and 87 near the carboxyl terminus (Fig.
1A). A second potential GAG-binding sequence, rich in basic residues, is located between residues 18 and 46. Unfortunately, the direct testing of heparin binding activity of apoSAA was hampered by the insolubility of delipidated apoSAA under physiological buffer conditions in the absence
of chaotropic agents (urea, SDS, and CHAPS). But two Met residues at
positions 16 and 23 facilitated the removal of the amino-terminal
amphipathic lipid binding domain (residues 1-24) (53) by CNBr cleavage
(Fig. 1A). For apoSAA1 this reaction generated an insoluble
16-mer (apoSAA1-16), and two soluble fragments, a 7-mer
(apoSAA117-23) and an 80-mer (apoSAA124-103) which constituted about 78% of the native protein. For apoSAA2 a
substitution of Ile with Met at residue 76 introduces an additional cleavage site allowing the 80-mer to be cleaved into a 53-mer (apoSAA224-76) and a 27-mer (apoSAA277-103)
(Fig. 1A). The GAG-binding consensus sequence was predicted
to lie within the 27-mer. Both apoSAA1 and apoSAA2 were purified (Fig.
1B), and cleavage of the individual isoforms with CNBr
yielded the expected fragments, with a minimum of secondary by-products
(Fig. 1, C and D). The identity of the
CNBr-generated peptides was determined by amino-terminal sequencing and
molecular weight analysis by mass spectrometry (data not shown).

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Fig. 1.
Mouse apoSAA1 and apoSAA2 isoforms and their
CNBr peptides. A, alignment of mouse apoSAA1 and
apoSAA2 showing their CNBr cleavage fragments, a 16-mer, 7-mer, and
80-mer for apoSAA1 and a 16-mer, 7-mer, 53-mer, and 27-mer for apoSAA2.
The basic residues (+) and the GAG-binding consensus sequence
(box) are shown, and dashed lines represent
apoSAA2 residues identical to apoSAA1. The apoSAA isoforms and their
respective CNBr-generated peptides were purified by RP-HPLC on a C18
Vydac semi-preparative column (see "Experimental Procedures").
B, apoSAA1 and apoSAA2 isoforms eluted at 27.2 and 29 min,
respectively. C, the apoSAA1 CNBr peptides, a 7-mer
(apoSAA117-23), an 80-mer (apoSAA124-103),
and a 16-mer (apoSAA11-16), eluted at 18.3, 25.6, and 29 min, respectively. D, the apoSAA2 CNBr peptides, a 7-mer
(apoSAA217-23), a 27-mer (apoSAA277-103), a
53-mer (apoSAA224-76), and a 16-mer
(apoSAA21-16), eluted at 18.2, 19.5, 26.4, and 30.6 min,
respectively.
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The Heparin-binding ApoSAA CNBr Peptides--
Preliminary
chromatographic analysis of the apoSAA1 and apoSAA2 CNBr cleavage
products on a heparin/Affi-Gel column (6 ml) revealed that both
preparations contained heparin binding activity (Fig.
2). The bound peptides were eluted from
the column by increasing the NaCl concentration, suggesting that the
association was primarily electrostatic in nature. The interactions
involved only heparin since binding was not detected with the uncharged
agarose Affi-Gel matrix alone (data not shown). Amino-terminal
sequencing of the heparin-bound peptides repurified by RP-HPLC
identified the peptides as apoSAA124-103 (m80-mer) and
apoSAA277-103 (m27-mer). The second potential GAG binding
region contained on the m53-mer did not to have heparin binding
activity. Further analysis of GAG binding activities was performed on
matrices produced by covalently coupling different GAGs to Affi-Gel or
Sepharose 4B and analyzed on a Waters HPLC apparatus. The two different
coupling reactions linking the GAGs through their accessible carboxyl
(Affi-Gel) or hydroxyl/amino groups (CNBr-Sepharose 4B) allowed us to
sterically block different GAG chain motifs. We found that
heparin-Sepharose 4B columns gave better resolution than the
commercially available heparin/Affi-Gel or the heparin/Affi-Gel 102 we
generated. The use of an HPLC apparatus also allowed for precise
solvent delivery and gradient formation, resulting in highly
reproducible retention times (±1-2%) and accurate determination of
NaCl concentrations at which peptide desorption took place.

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Fig. 2.
Screening of apoSAA1 and two CNBr peptides
for heparin binding activity. ApoSAA CNBr cleavage products were
dissolved in 20 mM Tris-HCl, pH 7.5, and loaded onto a 6-ml
heparin/Affi-Gel column pre-equilibrated with the same buffer ( ,
apoSAA1 CNBr peptides; , apoSAA2 CNBr peptides). The column was
washed at 30 ml/h for about 4 bed volumes and then developed with a 5 bed volume 0-1 M NaCl gradient, monitoring the eluant at
214 nm. The peptides contained in the different peaks were identified
by amino-terminal sequencing and molecular weight determination by mass
spectrophotometry.
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The Basic Amino Acid Residues Required for Heparin
Binding--
Since heparin binding activity was detected for the CNBr
27-mer peptide, we opted to identify the basic residues required for
binding using wild type and mutant synthetic peptides corresponding to
this 27-mer. Although our interest was initially drawn to the GAG-binding consensus sequence, an alignment of the mouse apoSAA sequence with 29 other apoSAAs from 12 different species indicated that
the consensus sequence was not conserved (Fig.
3A). Four of the basic
residues on this heparin-binding fragment were conserved, but only one
was part of the consensus sequence. Therefore, the role of all the
basic residues within this 27-mer was examined using nine synthetic
peptides, two wild type 27-mers corresponding to both the mouse
(m27-mer) and human apoSAA2 (h27-mer) and seven different mutant
peptides, five 27-mers in which basic residues, Arg-83, His-84, Arg-86,
Lys-89, and Arg-95, were replaced individually by Ala, one 27-mer in
which Arg-83, His-84, and Arg-86 were substituted simultaneously with
Ala, and a 20-mer missing the carboxyl-terminal Lys-102 (Fig.
3B).

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Fig. 3.
Protein sequence alignment of the
carboxyl-terminal 27 residues for apoSAAs. A, the
sequences for the carboxyl-terminal 27 residues for the apoSAAs were
retrieved from the GenBankTM data base (National Center of
Biotechnology Information) and compared with that of mouse apoSAA2
(m27-mer); the conserved basic residues are shown in bold;
dashes represent residues identical with the m27-mer, and
the GAG-binding consensus sequence is shaded. B,
synthetic peptides used to characterize the GAG-binding site, with the
Ala substitutions underlined.
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The synthetic version of the apoSAA277-103 peptide
(m27-mer) bound heparin-Sepharose 4B desorbing at 0.27 M
NaCl (RT = 40.4 min) (Fig.
4A). Although the columns were
normally washed with 3 bed volumes before starting the NaCl gradient,
washing for up to 20 bed volumes did not elute the peptide (data not
shown) indicating that it was immobilized with a partition coefficient (
) of
0.95, where the bed volume = 1/1
(54).
Purified apoSAA1 m80-mer peptide was also retained by the column
desorbing at 0.28 M NaCl (RT = 41.0 min) (Fig.
4A). The wild type peptides did not have an affinity for the
agarose matrix alone, Affi-Gel 102, or CNBr-activated Sepharose-4B
blocked with ethanolamine. As additional controls, carbonic anhydrase
and bovine serum albumin lacked binding activity for the heparin
columns, whereas fibronectin and LDL, known heparin-binding proteins,
both bound the heparin columns. The binding characteristics of all the
synthetic peptides were also tested on heparin/Affi-Gel and found to be
similar to heparin-Sepharose 4B with the exception of the
Lys-102
peptide (described below).

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Fig. 4.
Identification of the basic residues of the
m27-mer, within the GAG-binding consensus sequence (A)
and outside the GAG-binding consensus sequence (B),
which are important for heparin binding. Nine synthetic peptides
were applied to heparin-Sepharose 4B, the m27-mer, R83A, H84A, R86A,
R83A/H84A/R86A (triple mutant), K89A, R95A, Lys-102
(mouse apoSAA277-96) which lacked the carboxyl-terminal
Lys-102, and the h27-mer (human apoSAA278-104). The
elution profile of the Lys-102 peptide on
heparin/Affi-Gel is also shown. All peptides were chromatographed
separately, and their corresponding peaks are labeled on the graph.
Peptides (25-50 µg) were applied to a heparin-Sepharose 4B column (3 ml), pre-equilibrated with 20 mM Tris-HCl, pH 7.5, at 0.5 ml/min with continuous monitoring of the eluant at 214 nm. After
washing with 3 bed volumes, the column was develop with a 0-1
M NaCl linear concentration gradient (10 bed volumes). The
m80-mer was generated by CNBr cleavage of apoSAA1 and purified by
RP-HPLC. The peptides were run separately, and composite representative
elution profiles are shown. The minor peaks present between 35 and 40 min may represent small amounts of impurities present in the synthetic
peptides.
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The first series of mutant peptides were designed to investigated the
Cardin and Weintraub (43) proposed GAG-binding consensus sequence
XBBXBX, residues 82-87 of apoSAA
(Fig. 4A). Of the individual replacements, R86A showed the
greatest reduction in heparin-Sepharose 4B binding, eluting at 12.4 min
(0 M NaCl), followed by R83A which eluted at 17.4 min (0 M NaCl). H84A reduced heparin binding only slightly
(RT = 38.1 min, 0.24 M NaCl), and these replacements in combination (R83A/H84A/R86A) had a cumulative effect on the inhibition of binding (RT = 7.1 min). The second series of mutant peptides tested the involvement of Lys-89, Arg-95, and Lys-102 for
heparin binding (Fig. 4B). Peptides K89A and R95A eluted
from heparin-Sepharose at 16.6 (0 M NaCl) and 24.0 min (0 M NaCl), respectively, indicating impaired binding
activity. Deletion of the carboxyl-terminal residue Lys-102
(Lys-102
) caused only a minor reduction in heparin
binding activity with the Lys-102
peptide eluting at 37.9 min (0.23 M NaCl). Human apoSAA278-104 (h27-mer), which lacks a typical GAG-binding consensus sequence, bound
heparin with an avidity comparable to that seen with the m27-mer
(RT = 39.5 min, 0.26 M NaCl). HDL-apoSAA, isolated
from acute-phase mouse plasma, did not appear to have any affinity for
heparin (Fig. 4B) suggesting that the heparin-binding site was cryptic on HDL-associated apoSAA. By omitting NaCl in the starting
buffer, it was possible to rank the relative importance of all the
basic residues of m27-mer for heparin binding based on the retention
times (RT in min) of the different mutant peptides. Arg-86(12.4
min) > Lys-89(16.6 min) > Arg-83(17.4
min) > Arg-95(24.0 min) > Lys-102(37.9
min) ~ His-84(38.1 min) ~ m27-mer(40.4 min). The first four mutant peptides had very
low affinity for heparin eluting at 0 M NaCl. When the
carboxyl-linked heparin column (heparin-Affi-Gel) was used a similar
hierarchy was observed (data not shown), but Lys-102 became more
important for binding, with Lys-102
desorbing at
0.047 M NaCl (RT = 26.8 min) versus 0.23 M NaCl (RT = 37.9 min) on heparin-Sepharose 4B (Fig.
4B).
GAG Binding Specificity of ApoSAA--
The binding specificity of
the 27-mers was further investigated by affinity chromatography on a
series of columns charged with either heparan sulfate (HS), chondroitin
sulfate (CS), dermatan sulfate (DS), or hyaluronan (HA). Binding of the
wild type m27-mer (Fig. 5A)
and h27-mer (Fig. 5B) was not detected with CS, DS, or HA
columns. However, the 27-mers and the m80-mer peptide bound to
HS-Sepharose 4B, desorbing at approximately the same ionic strength as
that seen with heparin-Sepharose 4B. The Lys-102
peptide
was only slightly retained by HS (RT = 16.5 min), suggesting that
the Lys-102 residue contributed significantly to apoSAA-HS interactions. Since carboxyl-linked heparin showed a reduced avidity for Lys-102
, and HS has one-third the sulfate content of
heparin and thus a lower net charge, we were interested to see if the
carboxyl groups of HS played a more prominent role than those of
heparin in apoSAA interactions. Little or no binding was detected
between HS/Affi-Gel and the m27-mer, Lys-102
, and h27-mer
peptides. However, fibronectin (Fig. 5B) and LDL (not shown)
still bound to this matrix, indicating that only the HS domain(s)
containing the apoSAA-binding site(s) were blocked. The importance of
the HS-carboxyl groups for apoSAA binding was further demonstrated when
binding was destroyed after blocking carboxyl groups on HS-Sepharose 4B
with 32 mg of carbodiimide, equivalent to the amount used in the
Affi-Gel coupling reaction (Fig. 5B).

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Fig. 5.
The GAG binding specificity was established
for the m27-mer (A) and the h27-mer peptides
(B). Heparan sulfate (HS), chondroitin
sulfate (CS), dermatan sulfate (DS), and
hyaluronan (HA) were coupled to either Sepharose 4B or
Affi-Gel 102 (see "Experimental Procedures"). The elution profiles
of the m27-mer and h27-mer peptides on HA-Sepharose and DS-Sepharose
(asterisks) were very similar to that of CS-Sepharose and
for simplicity are not shown.
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Hierarchy of Mutant Peptide Binding to HS--
The m27-mer mutant
peptides were also tested for HS binding (elution profiles not shown),
and their relative importance was compared with that for heparin
binding (Fig. 6). Each individual substitution/deletion of the basic residues, except His-84, had a
greater impact on binding to HS than to heparin. The net reduction in
RT for HS-Sepharose 4B ranged from 28.2 min for R86A to 23.6 min for
Lys-102
. For heparin-Sepharose the range was greater,
28.0 min for R86A to 2.5 min for Lys-102
. The Lys-102 and
Arg-95 residues appeared to be much more important for HS than for
heparin binding.

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Fig. 6.
A comparison of the relative importance of
the basic residues of the m27-mer peptide for heparin and HS
binding. The same series of peptides as in Fig. 4 were analyzed on
an HS-Sepharose 4B column, and the net reduction in RTs (wild type
m27-mer RT minus mutant peptide RTs) was plotted comparing heparin and
HS-Sepharose 4B columns. The sequence of m27-mer is shown
above the graph. Open bars, heparin-sepharose;
closed bars, heparan sulfate-sepharose.
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Sulfonate Spacing Is Important for ApoSAA Binding--
Taurine
(2-aminoethylsulfonate), a cysteine catabolite, and Congo Red (sodium
diphenyldiazo-bis-
-naphthylaminesulfonate, CR), an acidic diazo dye
used to stain amyloid deposits, are mono- and disulfonate compounds,
respectively. The sulfonate groups in CR are spaced 19.3 Å apart (Fig.
7A). Both taurine and CR also have amino groups which facilitated their coupling to Sepharose 4B and
oriented their sulfonate groups away from the matrix surface. For
taurine-Sepharose 4B, the sulfonates would be randomly distributed, but
for CR-Sepharose 4B they would be organized in regularly spaced pairs.
m27-mer binding was only detected for CR-Sepharose and was retained
through electrostatic interactions since desorption required increased
NaCl concentration (Fig. 7B). Neither the
Lys-102
nor the R83A/H84A/R86A triple mutant peptides
bound to CR-Sepharose, consistent with the CR-m27-mer interaction being
dependent on basic residues. This also indicated that the spacing of
the anionic groups on CR was important for binding and possibly mimics
the sulfate spacing in the apoSAA-binding motif on HS.

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Fig. 7.
Congo Red m27-mer interactions illustrating
that sulfonate/sulfate spacing is important in HS:peptide
binding. A, representation of the chemical
structures for taurine and Congo Red. B, elution profiles of
the m27-mer, R83A/H84A/R86A, and Lys-102 peptides on
taurine and/or Congo Red-Sepharose 4B columns.
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GAG-HDL/HDL-ApoSAA Interactions; ApoA-I Binds HS--
HDL-apoSAA
from inflamed mouse plasma did not demonstrate any heparin binding
activity which suggested that the heparin-binding site is inactive when
apoSAA is associated with HDL (Fig. 8). However, HDL-apoSAA did bind to HS-Sepharose 4B and HS/Affi-Gel columns. To determine if apoSAA was solely responsible for
HDL-apoSAA-HS binding HDL from normal plasma, lacking acute-phase
apoSAA, was purified and tested for heparin/HS binding. Its binding
pattern was found to be identical to that of acute-phase HDL-apoSAA,
suggesting that in addition to apoSAA, other HDL apoproteins could also
contribute to the HDL-apoSAA-HS interactions (Fig. 8). ApoA-I, is the
most abundant apoprotein of HDL, and although apoSAA can comprise up to
80% of the apoproteins on acute-phase HDL-apoSAA, the remainder is
mostly apoA-I (55). ApoE is known to bind heparin/HS, but as a minor
component of both normal and acute-phase HDL-apoSAA (<1% of
HDL3 apoproteins) (56), it would be found on a small subfraction of HDL and is unlikely to be responsible for such a high
proportion of the HDL binding to the heparin/HS columns. Hence, apoA-I
was purified, its GAG binding activity was tested and found to parallel
that of HDL's, binding specifically to HS, and not to heparin or
chondroitin sulfate (data not shown). These results demonstrate that
HDL-HS and acute-phase HDL-apoSAA-HS interactions could be mediated
through apoA-I, with apoSAA possibly contributing to the latter
interaction.

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Fig. 8.
HDL, HDL-apoSAA (acute-phase), and apoA-I
binding activities on heparin-Sepharose 4B and HS-Sepharose 4B were
investigated and compared with the mouse apoSAA m80-mer peptide.
HDL (asterisks) was found to have very similar elution
profile to HDL-apoSAA and for simplicity is not shown here.
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DISCUSSION |
Heparin and heparan sulfate (HS) belong to a family of linear
heteropolysaccharides called glycosaminoglycans (GAGs),
which are normally synthesized linked to a protein core
(proteoglycans) (38, 39). The GAG chains consist of up to a hundred
disaccharide repeats composed of a hexuronic acid and a hexosamine. The
hexuronic acid is either
-D-glucuronic (GlcUA) acid or
the C-5 epimer
-L-iduronic acid (IdceA), and the
hexosamine is glucosamine (GlcN), with N- and
O-sulfation at various positions in the disaccharide
repeats. During its biosynthesis, heparin is extensively modified by a C-5 epimerase and three different sulfotransferases, producing a mature
chain in which the hexuronic acid is
80% IdceA and is almost
completely sulfated, with 2 to 3 sulfates per disaccharide unit. By
comparison, HS is less extensively modified (IdceA
50%) containing a
more varied structure with regions of high and low sulfation, 2 to 0.2 sulfates per disaccharide. Their distribution in tissue is also very
different, with heparin being confined to cytoplasmic granules of mast
cells, whereas HS is found ubiquitously on cell surfaces and within the
extracellular matrix. Given the limited anatomic distribution of
heparin, binding studies with HS may be the more relevant.
Due in part to their net negative charge, heparin/HS can associate with
a variety of proteins (57). These interactions have profound
physiologic and pathologic importance since they modulate the function
of numerous proteins such as growth factors, serine protease
inhibitors, extracellular matrix proteins, plasma lipoproteins, and
lipolytic enzymes. Among the apolipoproteins, apoE and apoB have well
characterized heparin/HS binding activities (58-61). To this list we
can add two other apolipoproteins, apoSAA and apoA-I. By affinity
chromatography of CNBr fragments and synthetic mutant peptides of
apoSAA, we were able to map the heparin/HS-binding site to the
carboxyl-terminal end for both mouse (residues 77-103) and human
(residues 78-104) apoSAA. The basic residues, Arg-83, Arg-86, Lys-89,
and Arg-95 of the mouse peptide (m27-mer) were found to be necessary to
maintain full heparin binding activity, and for HS binding Lys-102
residue was also required. The binding hierarchy of the basic residues
differed between heparin and HS possibly reflecting differences in GAG
ligand structure. Differences in basic residue composition and spacing
between heparin and HS binding sequences have also been observed by
others (62-64). Heparin-binding sequences tend to be richer in Arg
residues which are arranged in clusters. Arg residues have been shown
to have a higher affinity than Lys residues for heparin and sulfate
groups. For HS-binding sites, Arg residues are less common, and basic
residues are generally more widely spaced (64).
The other major GAGs (CS, DS, or HA) normally found in tissue showed no
detectable affinity for the apoSAA peptides. These data not only showed
that the GAG binding activity of apoSAA was specific for heparin/HS but
also provided clues as to the nature of the apoSAA-binding motif on HS.
The structures of DS and HA both contain elements resembling HS. HA is
composed of unsulfated GlcUA/GlcN units which are similar to a large
portion of the unmodified HS chain. The lack of m27-mer-HA binding
suggests that heparin/HS binding is probably dependent on IdceA and/or
sulfates. DS and CS have a higher sulfate content than HS suggesting
that apoSAA-HS binding was not dependent exclusively on the net
cationic charge of HS and that the appropriate spacing of the
carboxylates and sulfates is probably necessary.
The binding activities of heparin and HS were influenced by the type of
coupling reaction used to link them to the column matrix,
i.e. carboxyl linkage on Affi-Gel versus
hydroxyl/amino linkage on Sepharose 4B. For heparin/Affi-Gel only the
binding avidity of the Lys-102
peptide was reduced;
however, with HS/Affi-Gel neither the wild type m27-mer nor the
Lys-102
bound possibly because of a higher dependence on
HS carboxyls for binding. This interpretation was supported by the
observation that binding was inhibited after treating HS-Sepharose with
carbodiimide which blocks carboxyls (52). Alternatively, the
differences in binding between heparin/Affi-Gel and HS/Affi-Gel may be
explained by preferential coupling of the IdceA C-5 carboxyl to the
column matrix. If the apoSAA-binding site on heparin and HS is located in a region rich in IdceA, then heparin with its greater IdceA content
would be predicted to have a greater number of binding sites. Hence the
apoSAA-binding sites on heparin/Affi-Gel may be less likely to be
sterically blocked than those on HS/Affi-Gel. In order to understand
better this difference in binding affinities, identification of the
apoSAA-binding oligosaccharide on both heparin and HS is required.
An influential paper by Cardin and Weintraub in 1989 (43) identified
two consensus sequences, XBBBXXBBX and
XBBXBX (X, hydropathic; B, basic residues) by comparing 12 heparin-binding sequences from a series of heparin-binding proteins.
Later a third consensus sequence was reported by another group
(XBBXXBBBXXBBX) (44). For
mouse apoSAA an XBBXBX sequence at
residues 82-87 initially attracted our attention; however, inspection
of other available apoSAA sequences (from 12 species) revealed that
this consensus sequence was not conserved. There are many examples of
heparin/HS-binding protein or peptides that also lack these apparent
heparin-binding "consensus" sequences including antithrombin III,
residues 124-145 (65), platelet factor 4, residues 46-70 (66), apoE,
residues 202-242 (58), and basic fibroblast growth factor, residues
25-46 and 111-141 (67). This would suggest that the primary sequence alone cannot define the heparin/HS-binding site. Protein conformation may also play a role in placing critical basic residues into
energetically favorable positions juxtapositioning them with cationic
groups on the GAG chain. In support of this view, Margalit and
colleagues (45) have found that by comparing the spatial distribution
of the basic residues for 18 known heparin binding domains, for which three-dimensional structures were available, two basic residues were
always about 20 Å apart (20.3-23.5Å). In binding sites with an
-helical or
-strand conformation, the two basic residues were
separated by 13 and 7 residues, respectively, without a discernible consensus sequence. Furthermore, it was demonstrated that a distance of
20 Å between the two outermost basic residues of a heparin-binding site could accommodate docking of the binding site to a heparin pentasaccharide, the size sequence of heparin that binds to
antithrombin III (68).
The secondary structure for apoSAA is unknown, however, based on the
spacing of basic residues observed by Margalit et al. (45),
if the m27-mer conformation were in an
-helix then Lys-89/Lys-102 are at the correct distance apart (13 residues) and if in a
-strand then Arg-95/Lys-102 would have the appropriate spacing (7 residues). None of the four basic residues absolutely required for heparin binding
(Arg-83, Arg-86, Lys-89, and Arg-95) fit the 7 (
-strand) and 13 (
-helix) spacing pattern. Interestingly, an His or Arg residue is
found at position 93 in all species, except mouse, and it is possible
that Arg/His-93 functionally replaces the mouse Arg-83. The
Arg-86/Arg-93 or Arg-86/His-93 are separated by seven residues and
could be spaced correctly on a
-strand.
The binding characteristics of the apoSAA m27-mer with Congo Red (CR)
seems to agree with the findings of Margalit and co-workers (45). CR is
an acid diazo-disulfonate dye used as a diagnostic stain for amyloid
deposits (33) and has been reported to binding amyloid precursors and
block fibrillogenesis (34-37). We found that the m27-mer peptide bound
through ionic interactions to CR-Sepharose 4B. Peptides missing the
basic residues at either end of the m27-mer (Arg-83, Arg-86, or
Lys-102) lacked any CR binding activity. As a control, taurine, a
monosulfonate, linked to Sepharose 4B had little or no affinity for
m27-mer. A major difference between these two columns was that with
taurine-Sepharose 4B, the sulfonates were randomly distributed, whereas
for CR the sulfonates were organized into pairs 19.3 Å apart (69).
From a pathological perspective, the apoSAA-HS binding activity is
consistent with a view that most or all amyloid types are formed by an
initial fibrillogenic amyloid precursor-HS interaction (17, 19, 20). It
may also explain a number of previously reported observations
concerning AA amyloidogenesis, namely the co-localization of HS with AA
amyloid fibrils, the HS-induced
-sheet conformational change in
apoSAA2 (27), and the inhibition of AA amyloid by alipathic sulfonates
designed to simulate HS (31). In addition to apoSAA and apoA-I, a
mutant form of which can deposit as amyloid in the peripheral nervous
system (familial polyneuropathy) (70), heparin/HS binding activity has
been demonstrated for three other amyloid precursors, A
and
PP
(29, 71), prions (72, 73) and amylin (74, 75).
If the linear distance between the two outer basic residues of most
HS-binding sites are arranged in 20-Å intervals, as Margalit and
co-workers suggest (45), this information may prove useful in the
design of anti-amyloid drugs that would be effective for a variety of
amyloids. The effectiveness of CR in binding to the apoSAA
heparin/HS-binding site and preventing both AA (34) and prion amyloid
deposition in vivo (35-37) may be due to the spacing of its
sulfonates. This agent which potentially aligns with two basic residues
within the binding site allows electrostatic interactions to take place
blocking potential amyloidogenic interactions with HS. Further support
for this idea comes from a recent report in which the efficacy of
different polysulfonates for preventing AA amyloid was tested (31). Of
the polysulfonates examined, polyvinylsulfonate was found to be the
most effective at preventing AA amyloid formation in vivo.
Computer modeling of the energy-minimized structure for the repetitive
subunit of polyvinylsulfonate predicts the spacing of the sulfonates to
be about 5.4 Å, placing every fourth sulfonate approximately 20 Å apart (76).
To our knowledge HDL, or its major apoprotein apoA-I, has not
previously been reported to bind HS. The physiological significance of
the HDL-HS interaction is unclear at present. For low density and
remnant lipoproteins, HS proteoglycan facilitates their binding to the
cell surface, contributing to their processing by lipoprotein lipase
and hepatic lipase, and mediates receptor-lipoprotein internalization (77, 78). Cholesterol efflux is believed to require HDL contact with
the cell surface (79), and candidate protein receptors for apoA-I have
recently been identified (80-82). The hepatic lipase-mediated uptake
of HDL by hepatocytes has also been reported to be dependent on HS
proteoglycan (83), and binding of both HDL and acute-phase HDL-apoSAA
to macrophage could be destroyed by the removal of surface HS with
heparinase (84). Therefore, HDL may associate with cell surfaces
through an apoA-I-HS interaction possibly mediating its cholesterol
efflux activity or clearance from plasma. The physiologic significance
of the apoSAA-heparin interactions is also at present unknown. Most
heparin-binding proteins can also bind HS, which is believed to be the
natural ligand in most cases (85). Hence, the value of heparin may be
limited to locating HS-binding sites and may not be the best ligand for
the detailed molecular analysis of HS-binding sites. Interestingly,
HDL-apoSAA did not bind heparin suggesting the binding site is masked
when apoSAA is associated with lipid, a situation that has also been observed for one of the heparin-binding sites on apoE, residues 192-243 (86).
There is evidence that apoSAA-HS interactions maybe involved in a
number functions proposed for apoSAA. The HDL binding capacity of
macrophage has been shown to increase in the presence of apoSAA, possibly for the purpose of redirecting HDL-dependent
reverse cholesterol transport during the acute-phase response (12, 13). Saturable, high affinity interactions (Kd = 8 nM) between human apoSAA1
and neutrophils have also been
detected and may be responsible for the apoSAA-mediated inhibition of
the oxidative burst response by activated neutrophils (87, 88). The
binding to neutrophils could be blocked with a carboxyl-terminal
peptide of human apoSAA1
, residues 77-104, a region that shows high
homology to the corresponding human apoSAA2 (h27-mer) peptide
exhibiting HS binding activity. The apoSAA1
peptide is also missing
an Arg or Lys at position 83 (the residue present in the mouse apoSAA2 GAG-binding consensus sequence) which confirms that Arg-83 is not
required for HS binding of non-mouse apoSAAs. Human apoSAA1
has also
been reported to have monocyte and leukocyte chemoattracting activity
but only after its release from the HDL possibly by limited proteolysis
near the site of injury (89). This activity could be mediated by apoSAA
peptide-HS interaction on cell surfaces and the extracellular matrix.
Although the precise function of apoSAA has not yet been demonstrated,
the identification of an evolutionarily conserved HS binding domain
suggests that apoSAA-HS interactions on cell surfaces plays a role in
the execution of apoSAA function.