(Received for publication, April 1, 1996, and in revised form, September 10, 1996)
From the Department of Pathology, Queen's University, Syl and Molly Apps Research Center, Kingston General Hospital, Kingston, Ontario K7L 3N6, Canada
Serum amyloid A isoforms, apoSAA1 and apoSAA2, are acute-phase proteins of unknown function and can be precursors of amyloid AA peptides (AA) found in animal and human amyloid deposits. These deposits are often a complication of chronic inflammatory disorders and are associated with a local disturbance in basement membrane (BM). In the course of trying to understand the pathogenesis of this disease laminin, a major BM glycoprotein, has been discovered to bind saturably, and with high affinity to murine acute-phase apoSAA. This interaction involves a single class of binding sites, which are ionic in nature, conformation-dependent, and possibly involve sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on laminin. Collagen type IV also bound apoSAA but with lower affinity. Unexpectedly, no binding was detected for perlecan, a BM proteoglycan previously implicated in AA fibrillogenesis, although a low affinity interaction cannot be excluded. Entactin, another BM protein that functions to cross-link the BM matrix and is normally complexed with laminin, could inhibit laminin-apoSAA binding suggesting apoSAA does not bind to normal BM. Since laminin binds apoSAA with high affinity and has previously been shown to codeposit with AA amyloid fibrils, we postulate that laminin interacts with apoSAA and facilitates nucleation events leading to fibrillogenesis. This work also provides further support for the hypothesis that a disturbance in BM metabolism contributes to the genesis of amyloid. The specificity and avidity of the laminin-apoSAA interaction also implies that it may be a normal event occurring during the inflammatory process, which mediates one or more of the functions recently proposed for apoSAA.
Serum amyloid A proteins (apoSAAs)1 are encoded by an ancient, multigene family, which has been conserved for at least 200 million years (1). In mice four genes are actively expressed, producing one constitutive isoform (apoSAA4), one intermediate acute-phase isoform (apoSAA3), and two archetypical acute-phase isoforms (apoSAA1 and -2) (2). The latter two are composed of 103 residues differing by 9 substitutions (3) and make up about 95% of the plasma apoSAA, associated mainly (90%) with high density lipoprotein (HDL) (4, 5). During the acute phase of the inflammatory response, their synthesis is induced in the liver by cytokines (interleukin-1, interleukin-6, and tumor necrosis factor) with peak transcription rates attained within 3 h (6, 7), which leads to an increase in plasma concentration of up to 1000-fold (to approximately 1 mg/ml) within 18-24 h, declining to less than 50 µg/ml by 48 h (8). Since their production is stimulated by infection or tissue injury, it is widely accepted that apoSAA enhances host survival by either neutralizing the infectious agent or contributing to the repair process (2, 9).
ApoSAA and at least 16 other unrelated, normally nonfibrillar proteins are known to be precursors of different amyloid deposits. Amyloid is a generic term describing pathological accumulations of fibrillar proteinaceous deposits, which are associated with a number of disorders including Alzheimer's disease, rheumatoid arthritis, chronic hemodialysis, diabetes, and cancer (10, 11). These amyloid fibrils invade primarily the extracellular space of organs, where they can disrupt tissue architecture and function. Although the fibrillar proteins may differ in different diseases, amyloids have common tinctorial, ultrastructural, and compositional features.
All amyloids are composed of nonbranching fibrils (7-10 nm) with a
crossed -pleated sheet conformation, which stain with Congo Red and
exhibit red/green birefringence under polarized light. In many cases
mutations in precursor proteins (e.g.. transthyretin,
-protein precursor) have been linked to accelerated deposition, but
such mutations are not a prerequisite. In addition, abnormal proteolysis has also been thought to precede fibril formation, but
again in many cases this has been shown to be unnecessary. However, it
has been recognized that the basement membrane (BM) matrix is disrupted
near amyloid deposits (12, 13, 14), and in those amyloids that have been
investigated, one or more BM components appear to codeposit with
amyloid (15, 16, 17). Where analyzed, the accumulation of specific BM
protein mRNAs coincided with amyloid deposition in affected tissues
implying that the BM components found in amyloid deposits do not
originate from preexisting BM, but from de novo synthesis
(18, 19, 20). Once secreted it has been demonstrated both in
vivo and in vitro, that BM components can spontaneously
assemble to generate a BM matrix (21, 22), a process that seems to be
disrupted in and around amyloid deposits.
How apoSAA becomes deposited as an amyloid is still largely unknown. During reactive (secondary) amyloidosis, a complication of chronic inflammation, apoSAA is deposited in the spleen, kidney, and liver, where it is processed leaving the amino half to two-thirds of its sequence behind in the fibril (23). In mice only apoSAA2 serves as the amyloid precursor, and its amino-terminal sequence (1-15 residues), which differs from apoSAA1 by two residues, can form fibrils in vitro, albeit only at very low pH (24). For humans, six allelic apoSAA variants, produced by three SAA genes, show no difference in sequence at the amino end and may explain why they all have amyloid forming potential (2).
Significant advances in understanding the mechanism of amyloidogenesis
have emerged from immunohistochemical studies which have demonstrated
that BM components codeposit with amyloid (16, 25). In addition,
heparan sulfate (HS), a glycosaminoglycan covalently linked to the
BM-type proteoglycan, perlecan, has been shown to cause an increase in
-sheet structure (prevalent in amyloid) exclusively in apoSAA2 (26)
and not with apoSAA1, nor with apoSAACE/J isolated from an
amyloid-resistant mouse strain (23). For the Alzheimer's amyloid
peptide (A
), HS was found to enhance fibrillogenesis in
vitro (27). Recently, HS analogs (anionic polysulfates and
polysulfonates) have been shown to reduce AA amyloid accumulation
in vivo and also interfere with HS-induced A
fibril
formation in vitro (28). These observations support the
hypothesis that specific interactions between amyloid precursor proteins and BM components, particularly perlecan, may facilitate amyloid formation and cause the concurrent disruption in BM structure. As a first step to test this hypothesis, we investigated apoSAA's binding potential for the major BM components, collagen type IV (C-IV),
entactin (also called nidogen), laminin, and perlecan.
Employing an ELISA technique proven very effective in the
characterization of interactions involving different BM components (29, 30, 31), we have detected and characterized a saturable, high
affinity, association between laminin (laminin-1 from
Engelbreth-Holm-Swarm tumor) and murine apoSAA preparations
containing apoSAA1 and apoSAA2. Laminin-1 is the prototype of the
laminin family of BM glycoproteins composed of three different subunits
(1,
1, and
1) arranged into a multidomain cruciform structure
with three amino-terminal short arms and one carboxyl-terminal long
arm. Entactin, a BM protein that functions to cross-link the BM and is
normally complexed with laminin (32), could inhibit laminin-apoSAA
binding. In addition, apoSAA, like entactin, had no effect on the
Ca2+-dependent polymerization of laminin, an
integral process in BM assembly (22, 33, 34, 35). These observations
suggest that the apoSAA binding site(s) may map close to the entactin one identified on the laminin
-chain (36), and are clear of the
amino-terminal globular domains required for polymerization (35).
Perlecan binding of apoSAA was not detected, but this does not exclude
the possibility of a low affinity association.
The data presented suggest that apoSAA interacts in situ, with laminin which is free of entactin, and possibly in concert with HS facilitates apoSAA fibrillogenesis. We could find no evidence that the coincidental disturbance in BM organization observed with AA amyloidosis was caused by this interaction. Since the avidity of the laminin-apoSAA interaction is maximal under physiological conditions (pH, ionic strength) with "wild-type" proteins, we also postulate that it contributes to the normal acute phase response, possibly mediating some of the functions recently proposed for apoSAA.
Plasma apoSAA concentrations were experimentally elevated in CD1 mice by a subcutaneous injection of 0.5 ml of 2% (w/v) AgNO3 (37), resulting in a sterile abscess and an acute inflammatory state. After 18-20 h, mice were sacrificed by CO2 narcosis and exsanguinated by cardiac puncture preventing clotting with a small amount of 7% EDTA. High density lipoprotein containing apoSAA (HDLSAA) was isolated from plasma by sequential density flotation (38). The density of the plasma was adjusted to 1.063 g/ml with KBr or NaBr and centrifuged at 175,000 × g for 18 h in a 70.1Ti rotor (Beckman) at 4 °C. The top layer containing very low density lipoprotein/low density lipoprotein was removed and discarded. The pooled infranatants were adjusted to a density of 1.21 g/ml and re-centrifuged at 250,000 × g for 48 h at 4 °C. The top layer (HDLSAA) was aspirated, pooled, dialyzed against 0.15 M NaCl, 0.1% (w/v) EDTA, pH 6.4 (2 × 1 liter), for 18 h.
ApoSAA was purified from HDLSAA by dialysis against 10%
formic acid, pH 2.0 (2 liters), for 18 h at 4 °C, followed by
gel filtration on a Sephacryl-S-100HR column (2.5 cm × 110 cm)
eluted in the same buffer at 25 ml/h. The first major peak contained mainly apoA-I/A-II/C, and the second contained apoSAA1 and apoSAA2, which was collected, quick-frozen in liquid N2,
lyophilized, and stored at 20 °C.
AA peptides were purified from amyloid-laden mouse spleens by the method of Skinner et al., 1983 (39). All protein concentrations were determined by the DC protein assay (Bio-Rad). Lipid free bovine serum albumin (BSA) was used as the reference protein. Purity of the apolipoproteins and AA peptides was evaluated by SDS-urea-polyacrylamide gel electrophoresis (PAGE) stained with Coomassie Blue R-250 (40).
Purification of Laminin, Entactin, and PerlecanBM
components were purified from Engelbreth-Holm-Swarm (EHS) mouse sarcoma
propagated in nonlathyritic mice (Swiss Webster; Charles River
Laboratories, Montreal, Quebec, Canada), harvested at 2-4 cm, frozen
in N2(l), and stored at 70 °C. Purification of laminin
(laminin-1 isoform) and entactin was carried out as described by Ancsin
and Kisilevsky (41). Tumor was homogenized and washed twice in 3.4 M NaCl, 50 mM Tris, 2 mM EDTA, 1 mM N-ethylmaleimide, 1 mM
phenylmethylsulfonyl fluoride, pH 7.5, centrifuged at 10,000 × g for 15 min, discarding the supernatant on each occasion.
The residue was extracted in the same buffer but with 0.5 M
NaCl, for 4-8 h, then centrifuged as above. The supernatant was
precipitated with 30% saturated ammonium sulfate and the precipitate
redissolved and dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.5 (TBS). NaCl was increased to 1.7 M, precipitating collagen type IV, which was removed by
centrifugation. The supernatant was applied to a Bio-Gel A5m (2.5 cm × 120 cm) gel filtration column eluted with TBS. Fractions
containing the laminin-entactin complex were pooled, concentrated, and
dialyzed against TBS-2 M guanidine HCl. The dialysate was
applied to a Sephacryl-S400HR (2.5 cm × 140 cm) column eluted
with the same buffer. Laminin (850 kDa) (42) and entactin (148 kDa)
(43, 44) fractions were pooled, dialyzed against TBS, concentrated,
frozen in N2(l), and stored at
70 °C. Purification
steps were monitored by SDS-PAGE (45). Perlecan was purified as
described previously (46). C-IV and fibronectin were purchased from
Sigma.
An enzyme-linked immunosorbent assay technique was carried out to study the interaction between BM proteins, apoSAA, and AA peptides. Rabbit anti-perlecan was generated against EHS perlecan. Anti-laminin and anti-C-IV were purchased from Sigma, and anti-entactin from Upstate Biotechnologies Inc. Polystyrene microtiter plates (Immulon 4, Dyntech Laboratories) were coated with 100 µl of fibronectin (0.5 µg/ml), entactin (0.2 µg/ml), or collagen type IV (0.5 µg/ml) in 20 mM NaHCO3, pH 9.6. For apoSAA (0.8 µg/ml) and AA peptides (0.8 µg/ml), 4 M urea was included in the coating buffer to disaggregate soluble complexes. After overnight incubation at 4 °C, the plates were washed with 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 (TBS), then incubated with 1% BSA in TBS (150 µl) for 2 h at 37 °C to block residual hydrophobic surfaces. Plates were washed with TBS containing 0.05% (w/v) Tween 20 (TTBS), incubated with ligands at different concentrations in the same buffer, and left overnight at 4 °C to allow maximum binding. Different additives were included in the binding buffer as stated under "Results." Plates were washed again in TTBS and incubated with mouse anti-laminin IgG (Sigma) diluted 1:750 in TTBS, 0.1% BSA for 2 h at 37 °C, rewashed, then incubated in the same way with goat anti-rabbit IgG conjugated with alkaline phosphatase (Boehringer Mannheim) at a 1:500 dilution. After washing, bound IgG was detected by the addition of an alkaline substrate solution containing 2 mg/ml p-nitrophenyl phosphate, 0.1 mM ZnCl2, 1 mM MgCl2, and 100 mM glycine, pH 10.0. Plates were left at room temperature for 15-30 min, and the reaction was stopped with 50 µl of 2 M NaOH. The absorbance due to the released p-nitrophenol was measured at 405 nm with a Titertek Multiscan/MCC 340 (Flow Laboratories). Controls using BSA-coated wells were included in all experiments to which binding was negligible. Additional controls included the incubation of laminin with different binding buffers such as TTBS, TTBS plus 0.3 M NaCl, TTBS plus 2 M urea, TTBS plus 10 mM N-ethylmaleimide, and TTBS plus 1 mM dithiothreitol. These conditions had no significant effect on the sensitivity of the antibody detection of laminin. The amount of bound ligand was determined by subtracting the absorbance of wells coated only with BSA from those coated with the test protein. For direct quantitation, laminin standards were precoated onto wells on the same plates as the test proteins in order to generate standard curves. Coating efficiency of laminin, apoSAA, and AA peptides on the microtiter plates was 90-100% based on the amount of residual protein remaining in the coating buffer after incubation. Binding data was analyzed as described previously (47, 48), with a nonlinear curve fit program (SigmaPlot, Jandel Scientific) using Equation 1 for a one-binding site model with nonspecific binding or Equation 2 for a two binding-site model with nonspecific binding, where S is the proportionality constant for nonspecific binding and L is the laminin concentration.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Polymerization of laminin was assayed as described by Yurchenco et al. (34). Laminin (0.3 mg/ml), initially centrifuged to remove aggregates, was incubated for 4 h at 37 °C in TBS with 1 mM CaCl2 or 15 µM ZnCl2 or CaCl2/ZnCl2. A 10 and 20 M excess over laminin of apoSAA (43 µg/ml) and HDLSAA (670 µg/ml), respectively, was included in a series of tubes to test their effect on laminin polymerization. Molarity was based on a Mr = 850,000 for laminin (42), Mr(average) = 12,200 for apoSAAs (49) (apoSAA1 = 12,600, apoSAA2 = 11,800) and Mr = 200,000 with a protein composition of 50% for HDL/apoSAA (49, 50). After incubation, samples were centrifuged for 15 min, at 12,000 × g, and the polymerized fraction was calculated by subtracting the supernatant concentration from the total.
All BM components used
in these binding studies were extracted from mouse EHS tumor as
described previously, prepared by us or purchased from scientific
companies (see "Materials and Methods"). Proteins were evaluated by
SDS-PAGE (45) prior to use (data not shown). ApoSAA was isolated from
HDLSAA lipoprotein particles in inflamed mice, which
contained the constitutive apolipoproteins, apoA-I (23 kDa), apoA-II
(8720 kDa), apo-C (3500 kDa), plus the apoSAA isoforms apoSAA4 (14 kDa), apoSAA1 (12.6 kDa), and apoSAA2 (11.8 kDa) as seen by
SDS-urea-PAGE (Fig. 1). Formic acid-treated HDLSAA was resolved into two peaks on gel filtration. The
first peak contained most of the apolipoproteins (apoA-I, -II, and -C) and lipid eluting as a high molecular weight HDL remnants. The second
peak was composed of apoSAA1 and apoSAA2 and was used in the binding
experiments described herein (referred to as apoSAA for
simplicity).
Laminin and Collagen Type IV Bind SAA
A preliminary screen of
the major the BM components revealed that laminin had the highest
binding activity (Kd 2 nM) for
apoSAA preparations (Table I). Laminin also bound the AA
peptides isolated from AA amyloid laden spleens with similar affinity.
Little or no binding was detected between apoSAA and the
laminin-entactin complex, entactin, or perlecan. C-IV bound with a
Kd of about 14 nM. The negative result
with laminin-entactin complex suggested that entactin blocked
laminin-apoSAA binding sites, which was confirmed by competition assays
described below.
|
Laminin-entactin, laminin-entactin-C-IV, and perlecan-fibronectin binding experiments were included as controls testing both the normal binding activities of these BM components, as well as the accuracy of the assay. The dissociation constants observed (Kd = 1.6-2.5 nM) were all within expected values previously reported (29, 30, 31). For some experiments the binding maxima were also measured (see below), although the precise number of binding sites per molecule could not be obtained with this assay because neither the orientation nor the availability of binding sites could be determined.
Requirements for Optimal Laminin-ApoSAA BindingSince laminin
showed the highest binding activity for apoSAA, we focused our
attention on the physicochemical nature of this interaction.
Denaturation by heat had dramatically different effects on the two
proteins (Fig. 2). Laminin binding activity was
completely destroyed by heat denaturation (100 °C, 5 min), while
surprisingly, a similar treatment for apoSAA (in 20 mM
bicarbonate, 4 M urea, pH 9.6) appeared to increase
affinity 3-fold and binding maxima by a third (relative absorbance,
nanogram amounts not determined). Similar results were obtained with AA
peptides but with a more modest improvement in both affinity and
binding maxima (Fig. 2B).
Laminin-apoSAA binding activity was also influenced by a number of
common metals (Fig. 3, Table II). At
their respective normal plasma concentrations, ZnCl2 was
found to enhance laminin binding activity the most
(Kd = 1.8 nM,
Bmax = 20.1 ng), CuCl2 and
CdCl2 promoted binding to a lesser degree, while
MgCl2 and CaCl2 appeared to have an inhibitory
effect (compared with EDTA; Kd = 7.7 nM,
Bmax = 9.9 ng). The inhibition by
CaCl2 was not due to the promotion of
Ca2+-dependent polymerization of laminin since
both the temperature (4 °C) and laminin concentrations (up to 16 µg/ml) in this assay were well below the 37 °C and 0.1 mg/ml
critical laminin concentration required for polymerization to take
place (34). Trivalent metals such as Al3+ and
Fe3+ were found to cause a nonspecific increase in laminin
binding and are probably not important for this interaction (data not shown).
|
Binding activity was affected by the pH of the binding buffer (Fig.
4) with a change of as little as 0.5 pH units from pH 7.5 being enough to lower specific binding activity (Fig. 4). Neutral
to acidic pH levels had the greatest influence on binding maxima,
Bmax = 4.6 ng and 3.1 ng at pH 7.0 and pH 6.0, respectively, while affinity was affected most at alkaline pH levels:
Kd = 5.0 nM and 8.3 nM at pH
8.0 and pH 9.0, respectively. Nonspecific binding to BSA was not
changed over the pH range tested (data not shown). Hence laminin-apoSAA
binding appeared to be surface charge-dependent, although
protein conformation may have also been affected.
A number of compounds were also discovered to affect laminin-apoSAA
binding activity (Fig. 5, Table II). Chemical
denaturation with urea (2 M) prevented binding, confirming
that the interaction was protein conformation-dependent.
Increasing the NaCl concentration to 0.3 M also
significantly reduced binding consistent with the binding sites having
an ionic nature. Heparin blocked laminin-apoSAA binding most likely by
steric hindrance, occupying either one or more heparin binding sites on
laminin (51, 52), or the putative glycosaminoglycan binding site on
apoSAA (residues 82-87) (53).
Alkylation with N-ethylmaleimide (NEM) without reduction of
disulfide bonds also reduced the binding maxima by 79% without significantly affecting the Kd. This result
indicated that the apoSAA binding site(s) on laminin contains one or
more sulfhydryl groups (apoSAA has none). Laminin in fact has 42 Cys-rich repeats found on the amino-terminal ends of its three
subunits, of which 12 contained nested zinc finger consensus sequences
(41, 54, 55) (Fig. 6).
ApoSAA Affects Laminin Interactions Important for BM Assembly
Since the BM appears to be disorganized near AA amyloid
deposits, we investigated the possibility that laminin-apoSAA binding can disrupt key laminin interactions important for BM assembly. Two
such laminin interactions were investigated: one involving self-assembly forming homopolymers (21, 22), and the other a high
affinity interaction with entactin, another BM protein important for
the cross-linking of laminin and collagen type IV polymer networks (30,
56). Laminin polymerized in a Ca2+-dependent
manner as reported previously (33, 34, 35) unaffected by ZnCl2
(41) (Fig. 7). Neither apoSAA nor the HDLSAA
particle at 10 and 20 M excess of laminin, respectively,
inhibited laminin polymerization. However, apoSAA and entactin could
act as mutual competitors for laminin binding, suggesting their
respective binding sites colocalized (Fig. 8). Even at
the very low concentrations (picomolar) used in the ELISA, purified
laminin-entactin complex appeared to remained intact with little or no
laminin-apoSAA binding detected (Table I). When entactin was included
at a 5 M excess over laminin, binding to apoSAA was reduced
probably by a mixture of competitive and noncompetitive inhibition
since both affinity and binding maxima were significantly reduced (Fig.
8B, Table II).
ApoSAA was first discovered in the serum because of its
cross-reactivity with antisera against AA peptides isolated from AA amyloid (57, 58), and its deposition as amyloid appears to be an
aberrant consequence of the acute phase inflammatory response. Despite
more than 20 years of research since its discovery, the mechanism by
which soluble apoSAA becomes deposited as an insoluble fibrillar
structure has remained unknown. However, based mostly on work with
animal models, some pieces of the amyloid puzzle have been revealed. In
1985, Snow et al. (15) identified the major
glycosaminoglycan in amyloid deposits as heparan sulfate (HS), which
was subsequently shown to be linked to the protein core of the BM-type
proteoglycan, perlecan (15, 16, 17). The significance of this find was
further corroborated when of a number of different glycosaminoglycans
tested, HS alone increased the -sheet content for apoSAA2, the
amyloid precursor, but not for apoSAA1 nor apoSAACE/J, both
nonamyloidogenic proteins (23, 26). Later, it was observed that in fact
all the major BM components, including perlecan, codeposited with
amyloid.
From these results a hypothesis was put forth that one or more BM components may be involved in nucleation events leading to fibril formation. Such a mechanism would probably involve physical interactions with apoSAA, and after testing the major BM components, a saturable, high affinity association was detected between laminin (laminin-1 isoform), and an apoSAA preparation containing apoSAA1 and 2. This interaction involved a single class of binding sites, which appeared to be conformation-dependent, ionic in nature, and significantly enhanced by Zn2+. Heat or urea denaturation rendered laminin inactive. Boiling of apoSAA in 4 M urea, however, caused the binding activity to increase, indicating that the binding site on apoSAA may be a relatively short, peptide sequence made more accessible on denaturation of apoSAA.
The ionic nature of the interaction became apparent when the NaCl
concentration was increased or when the pH deviated from pH 7.5, both
resulting in decreased binding. The binding maxima was particularly
affected by lowering the pH to 7.0 and 6.0. In this pH range the
imidazole ring of His (pKa 6.0-7.0) may have
become protonated, gaining a positive charge and contributing to the
reduction in binding. Additionally, protonation of Cys sulfur atoms
would have reduced their ability to form a zinc finger (discussed
below). As the pH was increased to pH 8.0 and 9.0, binding affinity was
reduced without significantly affecting binding maxima. At these pH
levels, the Cys4-Zn2+ structure is probably
maintained, but a change in the ionization state of Lys and Tyr side
chains (pKa = 10.8 and 10.1, respectively) may have
taken place, causing the reduction in binding activity. These data
suggest that the laminin and/or apoSAA binding sites contain Cys and
His residues. However, one should bear in mind that, the binding
experiments were performed using intact proteins, and the ionization
state for the mentioned residues is influenced by their unique
microenvironments; hence, predicting the impact of these residues based
solely on the theoretical pKa of their individual
side chains is speculative. In addition, the binding sites on laminin
and apoSAA are likely affected simultaneously, and potentially in
different ways, further complicating the interpretation of the binding
data.
Since the region containing the first 15 residues of apoSAA is hydrophobic, and the binding activity for the AA peptides that are missing COOH-terminal 27-45 residues was similar to that for apoSAA, it is plausible that the laminin binding site is located between residues 15 and 76, a region in which ionizable residues are well represented. Experiments are presently being carried out to map the binding site within this region. Preliminary results suggest that apoSAA1 and apoSAA2 have common laminin binding sequences since both bound laminin equally well.2
In addition to being conformation-dependent, the apoSAA
binding site(s) on laminin may also require Zn2+ and free
sulfhydryls, as demonstrated by the stimulatory and inhibitory effects
of ZnCl2 and NEM, respectively. Similar effects were
observed for laminin binding of PP, the Alzheimer's amyloid precursor protein (48) and more recently, for laminin-entactin binding
(41). Incubation with a large excess of NEM (10 mM) was
found to reduced the Bmax of laminin-apoSAA
binding by 79%. The amino acid sequence in and around the remaining
binding sites may have protected them from modification by NEM.
Complete alkylation of laminin (reduce disulfide bonds prior to
alkylation) could not be tested, since reduction alone destroyed
binding activity virtually completely (Table II). Investigation of the
sequence for the entactin binding site, located on the laminin
-chain (36), revealed that it contained a zinc finger consensus
sequence (41). In fact, 12 potential zinc finger motifs within as many Cys-rich repeats could be found distributed on the laminin short arms,
and laminin-bound zinc (up to 8 mol/mol of laminin) has been detected
(41).
Like entactin, apoSAA had no effect on laminin polymerization, a
Ca2+-dependent process requiring the
amino-terminal globular domains distal to the Cys-rich repeats
(33, 34, 35). In addition, the ability of apoSAA and entactin to act as
mutual competitors for laminin binding indicated that their binding
sites may map close together. When the laminin-apoSAA binding activity
was assayed with entactin as the competitor, both the affinity and
binding maxima were reduced, reflecting either allosteric or a mixture of steric and allosteric inhibition. Therefore, one or more apoSAA binding sites must map some distance from the entactin site. However, we cannot exclude the possibility that one binding site also overlaps with the entactin one which would contribute to the reduction in
Bmax observed. By occupying its binding site of
no more than 58 residues on the laminin -chain (36), entactin as a
relatively large rod-shaped protein (148 kDa) probably exerts its
blocking effect over a wide region of the
and
-chain short arms.
There are six and three zinc finger-containing repeats on the
and
-chains, respectively, which might contain an apoSAA binding site or
sites. Taken altogether the data suggest that one or more, Zn2+-coordinated, zinc finger-like sequences may represent
the actual apoSAA binding sites, or at least contribute to them
significantly.
Based on their codeposition during amyloidosis (16) and saturable, high
affinity association in vitro, it is likely that laminin-apoSAA binding takes place at locations of amyloid deposition. Previous reports have provided strong evidence that perlecan through its HS side chains promotes fibrillogenesis (26), possibly by attaching
to a putative GAG binding site (residues 82-87) on apoSAA2 (53). High
affinity binding has been reported between perlecan and PP (47);
however, perlecan-apoSAA binding was not detected in this study. The
avidity of this interaction may be too low to have been measured by
ELISA but supports a role for laminin in apoSAA fibrillogenesis.
Specifically, this role may be to sequester apoSAA from plasma causing
its local concentrations to increase and/or provide a surface in close
proximity to HS, on which nucleation events could take place. Laminin
may also contribute directly to changes in apoSAA conformation favoring
fibril formation although this has not been investigated.
The aberrant synthesis of BM components may be a contributing factor in
amyloidogenesis. In brains of subjects with Alzheimer's, a significant
increase in laminin mRNA has been reported (18). In mice
experimentally induced to develop AA amyloid, increased levels of C-IV,
perlecan, laminin -chain, and entactin mRNAs are detected at
18-24 h, which precedes the histological accumulation of AA-fibril and
BM components (19, 20).3 Under normal
circumstances, newly secreted BM components would spontaneously
assemble through specific interactions to generate the BM matrix (21,
22) but at sites of amyloid depositions this process appears to be
blocked. We suspected that apoSAA might interfere with normal BM
assembly by inhibiting laminin's BM forming potential. However,
neither HDLSAA nor purified apoSAA affected laminin
polymerization, an integral step in BM assembly (21, 22). In addition,
apoSAA had little or no binding activity for laminin-entactin
complexes, the form in which laminin is secreted and incorporated into
BM by endothelial and other cell types (59, 60, 61). These complexes are
very stable (Kd
1 nM) and require
chaotropic agents such as guanidine HCl to be dissociated (32).
Hence it is unlikely that apoSAA could displace entactin from laminin, and the demonstrated ability of apoSAA to inhibit entactin binding of laminin may simply reflect the close proximity of their binding sites and not a dynamic exchange contributing to a disturbance in BM structure. The possibility that apoSAA may be influencing BM assembly through C-IV, for which it had a lower binding affinity, was not addressed in this study. However, laminin and C-IV homopolymer networks are believed to form independently (34), and a laminin-rich BM could be expected to form in the absence of C-IV polymerization. To our knowledge, widespread disruption in BM metabolism during inflammation has not been noted and BM homeostasis is probably not directly affected by apoSAA. Why BM synthesis would be associated with amyloid deposition in not known. The form in which laminin is secreted is also unknown, but our results suggest some laminin would have to be secreted free of entactin to allow apoSAA access to bind. This supports the idea that a disturbance in BM metabolism is linked, or even an underlying factor in amyloidogenesis.
The importance of the laminin-apoSAA interaction may not be solely pathological. Indeed the avidity of the laminin-apoSAA association suggests that apoSAA's function may be at the level of the BM. Additionally, because the apoSAA binding activity favors free laminin over the laminin-entactin complex, it is possible that abnormal or damaged BM may be the target of apoSAA. The rapid and transient appearance of apoSAA early in the injury/repair cascade is consistent with its binding to such BM soon after the injury has occurred, and probably not to newly synthesized BM which would appear later in the repair process, after most of the apoSAA is cleared from the plasma (62). Cellular release of proteases from damaged cells and activated neutrophils at injury foci would cause local damage to BMs, potentially exposing apoSAA binding sites on laminin. Entactin is known to be the most susceptible of all the BM proteins to proteolysis (63, 64). In contrast, the laminin short arms containing the Cys-rich repeats are remarkably protease-resistant (65, 66). Hence, it is conceivable that more laminin would be available for apoSAA binding at sites of injury.
Although the transcriptional and post-transcriptional regulation of acute phase apoSAA synthesis is well understood (67, 68, 69), the actual function of the proteins remains confused. Because a significant pool of apoSAA is located on HDL, which appears to enhance HDL binding to macrophages, it has been postulated that apoSAA focuses reverse-cholesterol transport (cholesterol transport from periphery to liver) to sites of injury, thereby optimizing removal of cholesterol from such sites (70). However, apoSAA has also recently been shown to increase secretory phospholipase A2 activity, which the authors suggested would lead to an increase in the ratio of HDL cholesterol-phospholipid, thereby promoting cholesterol delivery to sites of injury (71). Either scenario could be accommodated by apoSAA if it functioned to target HDLSAA particles to exposed laminin at sites of injury. Platelets use a similar strategy in response to injured blood vessels adhering to exposed BM via C-IV/laminin receptors (72, 73).
Some of the other functions proposed for apoSAA are not related to cholesterol metabolism and imply apoSAA is sequestered on HDL, possibly in an inactive state. These include inhibition of lymphocyte antibody response (74), the oxidative response in neutrophils (75), platelet activation (76, 77), and interleukin-1 and tumor necrosis factor-induced fever (78). ApoSAA has also been reported to block lymphocyte and metastasized tumor cell adhesion to BM (79), and to have monocyte and leukocyte chemoattracting activity (80, 81), both of which could be mediated through laminin-apoSAA binding. In the latter proposal apoSAA's chemotactic activity would be activated after release from the HDL, possibly by proteolysis, at or near sites of injury. This is analogous to the situation with complement factor C5, which is activated only after enzymatic cleavage by C5 convertase releasing the C5a peptide a chemoattractant and C5b a component of the membrane attack complex (82). As an amphipathic protein apoSAA contains putative lipid, calcium (83), glycosaminoglycan binding (53), and protein kinase C phosphorylation sites (84), plus a highly conserved domain unique to apoSAAs (2), and it is conceivable that these domains may contribute to different functions during inflammation.
We thank Dr. S. Narindrasorasak for supplying the mouse perlecan and rabbit anti-perlecan antibody.