(Received for publication, September 7, 1995; and in revised form, November 2, 1995)
From the
The specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I.
We show
that APP bound most efficiently to collagen type I in a
concentration-dependent and specific manner in the native and
heat-denatured states, suggesting an involvement of a contiguous
binding site on collagen. This binding site was identified on the
cyanogen bromide fragment 1(I)CB6 of collagen type I, which also
binds heparin. APP did not bind to collagen type I-heparin complexes,
which suggests that there are overlapping binding sites for heparin and
APP on collagen. We localized the site of APP that mediates collagen
binding within residues 448-465 of APP
, which are
encoded by the ubiquitously expressed APP exon 12, whereas the high
affinity heparin binding site of APP is located in exon 9. Since a
peptide encompassing this region binds to collagen type I and inhibits
APP-collagen type I binding in nanomolar concentrations, this region
may comprise the major part of the collagen type I binding site of APP.
Moreover, our data also indicate that the collagen binding site is
involved in APP-APP interaction that can be modulated by Zn(II) and
heparin. Taken together, the data suggest that the regulation of APP
binding to collagen type I by heparin occurs through the competitive
binding of heparin and APP to collagen.
The amyloid precursor protein (APP) ()belongs to a
gene family in which three genes are known. In addition to the APP
gene, the genes for the amyloid precursor-related proteins APLP1 and
APLP2 map to the human chromosomes 19 (APLP1) (1) and 11
(APLP2)(2, 3) . APP is encoded by the APP gene on the
long arm of human chromosome 21 and has attracted attention due to its
involvement in the deposition of amyloid
A4 protein in the brain
of patients with familial and sporadic Alzheimer's disease and
individuals with trisomy 21(4, 5) .
The role of APP
in the pathogenesis of Alzheimer's disease has been underscored
by the discovery of mutations within the A4 or sequences flanking
the
A4(6, 7, 8) . All of the identified
susceptibility genes linked to Alzheimer's disease appear to
influence the
A4 amyloid
formation(9, 10, 11) .
From the high degree of evolutionary conservation of the endo- and ectodomain of APP and its widespread tissue expression, APP has been expected to be implicated in a variety of cellular processes and events. Secreted isoforms of APP (APPs) containing a region homologous to the Kunitz protease inhibitor consensus sequence have a role in regulation of extracellular protease activity (12) and are endocytosed by the low density lipoprotein receptor-related protein(13) . A possible in vivo function is provided by the discovery that APP is a very potent inhibitor of factor XIa, and APP-factor XIa complexes might be involved in the regulation of the coagulation cascade(14, 15) .
Another possible function of the ectodomain of secreted or membrane-associated forms of APP have also been shown to be involved in neuronal-cell or cell-matrix interactions and cell growth regulation (16, 17, 18, 19) . Here, the ability of APP to stimulate cell adhesion and growth does not depend on the Kunitz protease inhibitor domain but may derive from its high affinities for heparin, heparan sulfate proteoglycans, laminin, and collagen type IV(19, 20, 21, 22, 23, 24, 25, 26) .
APP has been shown to bind Zn(II) and Cu(II) at two distinct sites (27, 28) and was recently found to belong to a family of zinc-modulated, heparin-binding proteins(29) . Zn(II) binding was shown to strengthen the binding of APP to heparin, thus demonstrating an interaction of residues involved in ligand binding which are located in different domains(21, 27) . Binding of metallic cations like Zn(II) and Cu(II) may control APP conformation and stability (27, 28, 30) and thus may promote the binding of APP to extracellular matrix elements like heparan sulfate proteoglycans(30) .
We report here the regulation of APP binding to collagen type I by heparin and the mapping of the binding sites for APP to collagen type I and vice versa. Our binding studies reveal that APP binding to collagen type I is mediated by residues 448-465. Since synthetic peptides representing this region show self-aggregational properties, it is suggested that APP-APP binding may exist. APP was identified in the human platelet to be present in membrane associated and soluble forms (31) , and collagen is particularly important for initiation of platelet activation leading to successful formation of a hemostatic plug(32) . For this reason our findings suggest that APP as an adhesive glycoprotein may participate in collagen-induced platelet aggregation and as APPs may take part in the regulation of the coagulation cascade at sites of vascular injury.
Different collagen types were dissolved in 10 mM NaOAc, pH 5.5, and diluted with 10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl, to appropriate concentrations just before use.
Protein concentrations were determined by amino acid analysis.
Collagen type I was dissolved at 10 mg/ml in 70% formic acid, and solid CNBr (Merck) was added to a final concentration of 20 mg/ml. Digestion was allowed to proceed for 18 h at 4 °C.
Peptides were synthesized according to published methods (33) and purified on C-18 columns with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Sequences were confirmed by amino acid sequence analysis (477A, Applied Biosystems).
A full-length
(Fd-APP) and truncated recombinant forms of human APP
(Fd-APP
, TP-APP
) were prepared and
purified in the form of a prokaryotic expressed Fd fusion protein by
methods essentially as described by Weidemann et
al.(35) . TP-APP
represents the secreted
form of APP
(
-secretase cleaved)(5) .
After preparative SDS-PAGE and electroelution, the soluble protein was separated from salts and SDS by Excellulose GF-5 columns (Pierce).
Protein concentrations were determined by amino acid analysis according to the manufacturer's protocol after hydrolysis with 6 N HCl, 0.1% phenol for 24 h at 110 °C (420A Amino Acid Analysis System, Applied Biosystems).
After adsorption of collagen to microtiter
wells, nonspecific binding sites were blocked by incubation for longer
than 1 h with 1% BSA in PBS. I-Labeled proteins were
diluted with PBS and added to the wells (1
10
to 2
10
cpm/well), together with the reagents to be
tested. After a 3-h incubation at room temperature, the wells were
emptied. The plate was divided into separate wells, and the
radioactivity of bound ligand was measured by liquid scintillation
counting.
The value obtained with wells coated with BSA was subtracted, as this represents nonspecific binding. Results were expressed as the mean of duplicate determinations, which usually did not differ by more than 10%.
Synthetic peptides representing
candidates for the collagen binding region on APP and control peptides
were dissolved in 0.1 PBS and tested as competing ligands for
I-APP-collagen type I (human placenta) binding. Collagen
type I was dot-blotted in duplicate onto nitrocellulose (0.05-10
µg) and nonspecific binding sites were blocked with 1% BSA in 1
PBS for 1 h at room temperature. The dot blot was cut into
strips and incubated with
I-APP together with and without
competing peptides for 3 h at room temperature in 0.1
PBS.
After incubation the dot blot was washed one time with blocking buffer,
and the dots were excised, placed in scintillant, and assayed by
counting. Variations of this binding assay were occasionally used and
are indicated in the text and figure legends.
CNBr cleavage of
isolated chains was performed as described(36) . The identity
of the 1(I)CB6 fragment has been confirmed by
NH
-terminal sequencing.
To determine the amount of collagen adsorbed to microtiter wells, 5 µg of each collagen type were allowed to adsorb in microtiter wells overnight followed by a brief rinse. Then protein was extracted from microtiter wells with three successive rinses of 100 µl of formic acid and subjected to amino acid analysis after hydrolysis. The amount of protein adsorbed to the microtiter wells was quantified and calculated to be 50% of the amount of that was loaded.
First, we tested which different collagen types interact with APP. A
dot blot assay revealed that I-labeled rn-APP binding to
fibrillar collagen type I and basement membrane collagen type IV (38) was saturable and specific. APP bound most efficiently to
collagen type I (data not shown).
To investigate the kinetics of APP binding to collagen types I and IV and to gelatin in more detail, we used two distinct assays. Radiolabeled rn-APP was incubated in microtiter wells with immobilized collagen types I and IV at room temperature in microtiter wells or BSA as a control. APP-gelatin interaction was tested by incubating the protein with gelatin-Sepharose beads.
The binding efficiency was observed to depend on salt
concentration and showed substrate specificity (binding to collagens,
but not to other extracellular matrix components, such as fibronectin
or vitronectin). At the specific activity used, 10% of the I-labeled rn-APP added bound to collagen type I in a dot
blot assay. Binding was saturable and analyzed by using the methodology
of Scatchard(39) . Assuming the presence of only one binding
site on the collagen types I and IV, K
values in
the range of 0.5
10
M for collagen
type I (Fig. 1), 4.5
10
M for collagen type IV, and 1.5
10
for
gelatin (data not shown) were calculated for the binding of APP to
native and heat-denatured collagen (gelatin).
Figure 1:
Binding curve of collagen type I with I-rn-APP. The protein-coated wells were incubated with
I-rn-APP at various concentrations. The inset indicates the Scatchard plot for the binding
curve.
Figure 2:
HPLC
purification of a putative collagen binding peptide obtained by
endoproteinase Lys-C digestion of Fd-APP encoded by exons
1-18 of APP (A) and Fd-APP
encoded by
exons 1-6 of APP (B). Only Fd-APP
yielded
the collagen binding peptide (black bar) determined by amino
acid sequence analysis. Other fractions (hatched area) were
revealed to contain collagen type I chains or fragments
thereof.
To confirm that we
identified the collagen binding site of APP, three peptides of residues
448-465, 448-480, and 471-493 (Table 1) were
synthesized to perform inhibition studies. Two of these peptides were
found to inhibit strongly the binding of I-hs-APP to
collagen type I in a dot blot assay, with a calculated IC
value of 150 nM (CBP1) and 75 nM (CBP) (Fig. 3), whereas control peptides did not have any influence at
concentrations of up to 20 µg/ml. As an independent control we used
iodinated NCAM
purified from rat brain according to
Probstmeier et al. (40) and did not observe any
inhibition of NCAM binding to collagen in the presence of CBP
concentrations of up to 5 µg/ml (data not shown).
Figure 3:
Dose-dependent inhibition of the binding
of I-hs-APP to collagen by the synthetic peptides CBP
(diamonds), CBP1 (open squares), and CBP2 (filled squares). The values
represent residual binding determined in dependence of the
concentration of the synthetic peptides as the competing ligands in a
dot blot assay.
Since APP
belongs to a superfamily, we further investigated synthetic peptides
that represent homologous sequences of CBP in the APLP to inhibit APP
binding to collagen type I. APP-collagen type I interaction could be
less effectively inhibited by synthetic peptides of mouse APLP1
(residues 445-477) (1) and human ALPL2 (2, 3) (residues 522-554). For both peptides,
inhibition was found to be dose-dependent with an IC of
about 1 µM (5 µg/ml) in a dot blot assay (data not
shown). But mouse APLP1 and human APLP2 peptides (Table 2) were
still able to inhibit binding of APP to collagen type I although with a
reduced capacity by a factor of 10. A possible explanation for this is
given by the secondary structure prediction for CBP/hs-APP,
CBP/mm-APLP1, and CBP/hs-APLP2 according to Chou and Fasman (Table 3; (41) ) that changes from
-structure for
the NH
terminus of CBP/APP to
-structure in the same
site of APLPs (Table 3).
Furthermore, direct binding of peptides CBP and CBP1 to collagen type I was proven by surface plasmon resonance (BIAcore, Pharmacia Biotech Inc.). Collagen type I was immobilized to the dextran surface of the sensorchip(21) , but the kinetics of the binding reaction could not be determined, because two independent binding events, CBP to collagen and CBP to CBP, were observed to superimpose. This led us to the conclusion that both peptides showed a strong tendency for self-aggregation and suggested that CBP could represent a binding site for APP.
If APP-APP binding
could occur through the CBP sequence, APP should bind specifically to
the synthetic peptide CBP. The binding was analyzed in a solid-phase
assay. I-rn-APP was added to microtiter plates coated
with the CBP in the absence or presence of Zn(II) and heparin. Binding
of APP was found to be saturable and dependent of the amount of CBP
coated to the microtiter plate (Fig. 4A). A
dose-dependent inhibition of the binding of
I-hs-APP to
CBP by TP-APP
was observed in nanomolar concentrations (Fig. 4B).
Figure 4:
Modulation of I-rn-APP
binding to the CBP peptide. No inhibitor (diamonds), heparin (open squares), and Zn(II) (filled triangles) were
added at concentrations of 100 µM ZnCl
and 10
µg/ml heparin. APP binding to CBP was found to be inhibited by
heparin and the divalent metal ions Zn(II) and Cu(II) in a
concentration-dependent manner (A and C). A
dose-dependent inhibition of the binding of
I-hs-APP to
CBP by recombinant APP was observed in nanomolar concentrations (B). Inhibition of
I-hs-APP binding to CBP
yielded for instance 70% at 1 µM Zn(II), 85% at 25
µM, and 90% at 50 µM Zn(II), but Ni(II),
Co(II), and Fe(III) were unable to inhibit (C).
The inhibition of binding was also found to be concentration-dependent for Zn(II) and Cu(II) but not for other divalent ions such as Fe(II), Co(II), and Ni(II) (Fig. 4C). Zinc(II) and copper(II) reduced binding to 10% (Zn(II)) and 20% (Cu(II)) at 50 µM, 15% (Zn(II)) and 35% (Cu(II)) at 25 µM, 30% (Zn(II)) and 50% (Cu(II)) at 1 µM concentrations. These values fall within the physiological Zn(II) and Cu(II) concentrations(42, 43) .
When we tested the effect
of Zn(II) (27, 30, 44) on binding of I-hs-APP to collagen type I, it did not have any effect
on APP collagen type I binding up to concentrations of 100 µM Zn(II) (data not shown), exceeding the saturating concentration of
Zn(II) binding to APP(27) .
Thus, under our experimental in vitro conditions, APP binding to collagen type I and CBP binding of APP are two mutually exclusive events of which only the binding of APP to CBP is regulated by the divalent metal ions Zn(II) and Cu(II) that both are ligands of APP(27, 28) .
Since it has previously been reported that APP-collagen interaction is mediated by a heparin bridge mechanism(23) , we investigated APP collagen type I binding in the presence of heparin and chondroitin sulfate.
Figure 5:
Dose-dependent inhibition of the binding
of I-rn-APP to collagen by heparin and chondroitin
sulfate. The binding to collagen type I immobilized on a microtiter
well was determined in the presence of increasing concentrations of
heparin and chondroitin sulfate and found to be
concentration-dependent.
Figure 6:
The 1(I)CB6 peptide binds
S-heparin and
I-rn-APP. The resulting
peptides of CNBr digestion of
1(I) and
2(I) chains were
fractionated by heparin-Sepharose chromatography. Fractions eluting at
600 mM NaCl were analyzed by SDS-PAGE (gradient from 10 to 15%
polyacrylamide) and stained with Coomassie (A) or incubated
with
S-heparin after blotting to nitrocellulose (B). Partially digested dimers and trimers of the
-chains, the
1(I)CB8, and the
1(I)CB6 fragment (arrow) bind
S-heparin. CNBr fragments of
1(I) and
2(I) chains were separated by SDS-PAGE and incubated
with
I-rn-APP after blotting to nitrocellulose. Binding
was found to the
1(I)CB6 fragment as the only CNBr fragment (C) indicated by the arrow. The asterisk indicates incompletely digested and/or covalently cross-linked
dimers of the
-chains.
In an overlay assay I-rn-APP
binding to collagen type I was observed to partially digested dimers or
trimers of the
-chains and to the
1(I)CB6 fragment as the
only CNBr fragment showing up at an apparent molecular mass of 25 kDa (Fig. 6C). To determine the identity of this fragment,
it was purified from SDS gels and characterized by amino acid sequence
analysis. Higher bands represent uncomplete digested and/or covalently
cross-linked dimers of the
-chains. The results presented above
show that the
1(I)CB6 fragment of collagen type I contains binding
sites for both APP and the glycosaminoglycan heparin.
Since it has
been indicated that the triple helical structure of collagen is
important for the interaction with heparin(46) , the 1 and
2 collagen chains and the CNBr fragment
1(I)CB6 were
separated by SDS-PAGE, electroeluted, and assayed separately in a dot
blot apparatus by incubation with
I-rn-APP. Binding was
strongest to the
1 chain of collagen type I, less binding was
observed to the
1CB6 fragment, and no specific binding could be
observed to the
2 chain of collagen type I (Fig. 7, A and B). This result is consistent with binding studies
for which
I-rn-APP and gelatin-Sepharose affinity
chromatography indicated that APP recognizes heat-denatured collagen as
well as triple-helical fibrillar collagen. Apparently, the APP binding
site in the
1CB6 fragment has less tight specificity, probably due
to the change of conformation of surrounding residues.
Figure 7:
Binding of I-APP to collagen
type I in a dot blot assay. No specific binding to the
2(I) chain
of collagen could be observed (0% of total was specific) (A).
Saturable and specific binding was found to the
1(I) chain (54% of
total was specific) (A), collagen type I (66% of total was
specific), and the CNBr fragment
1(I)CB6 (70% of total was
specific) (B). The total amount of protein immobilized to
nitrocellulose was determined by amino acid analysis. Numbering of
cyanogen bromide peptides (CB) of the
1 chain of collagen type I
according to Miller et al.(71) and Piez et
al.(72) .
In
conclusion, our observations reveal a mechanism of APP binding to
collagen that is regulated by glycosaminoglycans that compete with APP
for an overlapping binding site of the 1 CB6 fragment of collagen.
Although both components can interact with
glycosaminoglycans(21, 47) , our assay system allowed
us to determine that inhibition of APP binding to collagen by heparin
occurred on the collagen side by binding of heparin to collagen.
Our results show that APP can interact with collagen types I and IV, and this interaction can be influenced by glycosaminoglycans. The binding of APP to collagen shows substrate specificity, is saturable, and Scatchard plot analysis suggests one binding site for collagen type I. Substantially reduced binding to denatured collagen is not observed, a finding that is in common with fibronectin binding to collagens (48) .
In this study we concentrated on collagen
type I that showed the highest affinity of those that were tested. The
affinity is in good agreement with the K values
for the binding of J1 glycoproteins and NCAM to
collagens(40, 49) .
Collagen binding of APP was mapped to the carbohydrate domain of APP corresponding to amino acid residues 448-465, not including the carbohydrate attachment site at asparagine in position 467 (Table 1). The sequence of peptide CBP1 is conserved in rat, mouse, and human APP. A search in the ``Swiss-Prot'' Protein Data Bank did not reveal any significant homology to the CBP1 sequence in the known collagen-binding proteins such as fibronectin(50) , heat shock protein HSP47(51) , or bovine propolypeptide of von Willebrand factor (52) at the level of primary structure.
The inhibitory
effect exerted through peptides CBP and CBP1 on APP collagen binding
must be taken as an indication. Direct binding of these synthetic
peptides to collagen did occur, but could not be measured in terms of K values, because the tendency for
self-aggregation of CBP and CBP1 observed by surface plasmon resonance
as an undirected binding interfered with those measurements. The
sequence of CBP is predicted to adopt a
-strand conformation
followed by the beginning of a
-turn structure at residue 466,
whereas CBP derived from APLP1 and APLP2 may adopt an
-helical
structure. This might explain why sequences corresponding to the
collagen binding site of other members of the APP gene family were
still able to inhibit binding, but the capacity to compete was
drastically reduced in comparison with APP-CBP binding by more than a
factor of 10. Additionally, peptides containing a
-strand followed
by a
-turn are reported to be capable of undergoing isologous
interaction in an antiparallel manner(53) . For instance the
homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium discoideum has been mapped to an octapeptide
sequence YKLNVNDS in the NH
-terminal globular domain (53, 54) and is known to mediate cell-cell adhesion
via homophilic interaction. It is remarkable that the
-turn
structure in gp80 is also induced by a potential glycosylation
attachment sequence NXS(55) , as is the case for the
CBP sequence of APP. Although APP contains at this position the only N-linked carbohydrate chain(56, 57) ,
according to our experiments it seems unlikely that the carbohydrate
moiety is a regulator of collagen/gelatin binding activity, because
deglycosylated rat brain APP retained the ability to bind to collagen
as well as fusion proteins of APP expressed in bacteria. But it still
remains possible that the carbohydrate moiety induces a certain
conformation of APP.
The observed self-aggregation of CBP led us to postulate the binding of radiolabeled rat brain APP to CBP that we analyzed in a solid-phase binding assay. We found binding to be saturable and recombinant APP, Zn(II), and heparin to be able to inhibit. This provides evidence for APP-APP binding involving the AAQIRSQVMTHLRVIYER sequence. We were previously able to show that an allosteric effect on APP binding to heparin is caused by Zn(II)(21) , but not by other divalent ions. Moreover, we observed that the electrophoretic mobility of CBP aggregates in capillary electrophoresis is dependent on the salt concentration. Therefore we conclude that binding of APP to CBP is driven by ionic forces, and separately from this, we propose a regulation by the divalent ions Zn(II) and Cu(II). We had shown earlier, in the case of Cu(II), that the modulation is likely due to the formation of APP aggregates and thus inhibiting the binding to the peptide on the solid support(28) .
Our assay system allowed us to discern that
inhibition of APP binding to collagen type I occurs on the APP or the
collagen side, although both components can interact with
glycosaminoglycans(47, 58) . We could demonstrate that
APP interacts directly with the 1(I)CB6 fragment of collagen type
I (Fig. 6C and Fig. 7B), independent
from integrin-mediated adhesion. Integrins bind to
1(I)CB3
(integrin (
2
1)) and
1(I)CB8 (integrin (
1
1))
fragments of collagen(59, 60) .
This interaction is regulated by heparin, and therefore both molecules probably share the same binding site on collagen. Heparin that is believed to act in vivo as a specific regulator of the structure and function of basement membranes(61) , or local variation in the concentration of heparin-like macromolecules, might be crucial for the interaction of APP and collagens in vivo.
A functional
collagen binding site of APP is of great importance, since
transmembrane APP could serve directly as a cell adhesion molecule, and
the secreted forms might be involved in the regulation of cell
interactions. In the periphery, APP might act as a
transmembrane-signaling molecule. For instance platelets are activated
by agonists such as thrombin and collagen (14, 15, 31) followed by the secretion of
APP, which is an abundant platelet -granule protein. It should be
mentioned that platelets contain 20-50-fold higher concentrations
of Zn(II) than plasma, and upon activation, platelets release Zn(II) to
the external platelet surface(62) . At the microenvironment of
the activated platelet in higher local concentrations, Zn(II) could
shift the properties of APP into a different site of action, i.e. the regulation of the coagulation cascade by inhibiting factor
XIa(63) .
Collagen can also act as an effective substrate
for neurite outgrowth from peripheral neurons and also promote
outgrowth from at least some central neurons(64) . An
involvement of APP in those processes was earlier suggested by Breen et al.(23) who found cell binding to a collagen
substrate (neuron-neuron and neuron-glia binding) inhibited by
antibodies against the extracellular portion of APP. Neuronal
regeneration is associated with the expression of extracellular matrix
proteins(65, 66) . A direct interaction of mature
transmembrane APP with components of the extracellular matrix might
reflect the importance of APP as a neuronal cell adhesion molecule. The
interaction of APP with extracellular matrix molecules may turn out to
have a role in the pathogenesis of Alzheimer's disease.
Proteoglycans appear to be increased in the vicinity of A4 amyloid
plaque deposition(67, 68) . Collagen-like molecules
such as the A12 form of acetylcholinesterase are also increased in the
areas of amyloid. Alterations in the affinity of APP for extracellular
matrix molecules by zinc may also be relevant to Alzheimer's
disease(21, 27) .
The normal processing of APP in the central nervous system may therefore be affected by APP-matrix interactions and deserves closer scrutiny.