(Received for publication, August 15, 1995)
From the
The serpin-enzyme complex (SEC) receptor was originally
identified using a synthetic peptide (peptide 105Y) based on the
sequence of a candidate receptor-binding domain of 1-antitrypsin
(1-AT) and was subsequently shown to be a receptor on the surface of
hepatocytes, monocytes, and neutrophils for recognition of
1-AT-elastase and several other serpin-enzyme complexes
(Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and
Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,
3753-3757). Studies of the minimal requirements for binding to
SEC receptor (SEC-R) showed that a pentapeptide FVFLM within the
carboxyl-terminal tail of
1-AT was sufficient for binding to SEC-R
and interacted with SEC-R in a sequence-specific manner (Joslin, G.,
Krause, J. E., Hershey, A. D., Adams, S. P., Fallon, R. J., and
Perlmutter, D. H.(1991) J. Biol. Chem. 266,
21897-21902). Sequence motifs bearing homology with this
pentapeptide domain were found in the amyloid-
peptide, and
amyloid-
peptide 1-42 was shown to compete for binding to
SEC-R on hepatoma cells (Joslin, G., Fallon, R. J., Bullock, J., Adams,
S. P., and Perlmutter, D. H.(1991) J. Biol. Chem. 266,
11281-11288). In this study we examined the sequence specificity
by which amyloid-
peptide competes for binding to SEC-R and
examined the possibility that SEC-R is expressed in cells of neuronal
origin. The results show that amyloid-
-(25-35) and
amyloid-
-(31-35) compete for binding to SEC-R as effectively
as amyloid-
-(1-39), amyloid-
-(1-40), and
amyloid-
-(1-42). Amyloid-
-(1-16) does not compete
for binding to SEC-R. There is cross-competition for binding to the
same site by
I-peptide 105Y and
amyloid-
-(25-35) as well as by
I-Y
amyloid-
-(25-35) and peptide 105Y. By deletions and
substitutions within amyloid-
-(25-35) and generation of
chimeric amyloid-
-
1-AT peptides, amyloid-
-(31-35)
is shown to be critical for binding to the SEC receptor. However, the
upstream region, amyloid-
-(25-30), also contributes to
recognition by SEC-R. The SEC-R is present on the surface of a neuronal
cell line PC12 as well as that of murine cortical neurons in primary
culture, and the specificity of neuronal SEC-R for amyloid-
peptide is identical to that on hepatoma cells. Finally, SEC-R mediates
internalization and degradation of amyloid
-peptide in PC12 cells.
These results provide evidence that SEC-R plays a role in metabolism of
amyloid-
peptide in the nervous system.
The serpin-enzyme complex receptor (SEC-R) ()is a
cell surface protein of hepatoma cells, mononuclear phagocytes, and
neutrophils that has a ligand binding subunit of
70-78 kDa
(reviewed in (1) ). It was originally identified as the
receptor responsible for feed-forward regulation of
1-AT synthesis
by complexes of neutrophil elastase and
1-AT. A synthetic peptide,
peptide 105Y (SIPPEVKFNKPFVYLI), based on the sequence of a candidate
receptor-binding domain in the carboxyl-terminal tail of
1-AT
itself, was shown to mediate increases in synthesis of
1-AT in
human monocytes and human hepatoma HepG2 cells(2) .
Radioiodinated peptide 105Y bound to HepG2 cells with specific,
saturable, and reversible characteristics. Scatchard analysis predicted
a k
of
40 nM and
450,000 plasma membrane receptors/cell(2) . Binding of
I-peptide 105Y to HepG2 cells was blocked by unlabeled
elastase-
1-AT, cathepsin G-
1-antichymotrypsin,
thrombin-antithrombin III, thrombin-heparin cofactor II, and, to a
lesser extent, C1s-C1 inhibitor and tissue plasminogen
activator-plasminogen activator inhibitor I complexes but not by the
corresponding native proteins(2, 3) . Binding of
radioiodinated elastase-
1-AT, cathepsin G-
1-antichymotrypsin,
thrombin-antithrombin III, thrombin-thrombin-heparin cofactor II, and
tissue plasminogen activator-plasminogen activator inhibitor I to HepG2
cells was also partially, but specifically, blocked by peptide
105Y(2, 3) . Other studies have shown that SEC-R
mediates endocytosis and intracellular catabolism of elastase-
1-AT
complexes in HepG2 cells (4) and mediates directed migration
of neutrophils toward
1-AT-elastase complexes(5) .
In
studies designed to determine the minimal requirements for binding to
SEC-R, a series of smaller peptides based on the sequence of peptide
105Y were synthesized(6) . The studies showed that the
carboxyl-terminal pentapeptide FVYLI and its counterpart in the native
1-AT sequence, FVFLM, were sufficient for binding to SEC-R and
that interaction between these pentapeptides and SEC-R was
sequence-specific. A search for homologous sequences in other
eukaryotic proteins showed similarities in the carboxyl-terminal
regions of substance P, several other tachykinins, bombesin, and
amyloid-
peptide(6) . In fact, the homologous region of
amyloid-
peptide, amyloid-
-(25-35), is toxic to
neurons(7) . It constitutes the minimal peptide sequence for
the neurotoxic effect of amyloid-
-(1-42) (7) and,
therefore, has been considered a possible common final pathway for the
neuronal degeneration in Alzheimer's disease. Studies in HepG2
cells have subsequently shown that amyloid-
peptide competes for
binding of radioiodinated peptide 105Y and radioiodinated
elastase-
1-AT complexes to SEC-R(8) . Moreover, binding of
amyloid-
peptide to SEC-R in neutrophils results in chemotactic
activity and confers homologous desensitization to the chemotactic
activity of peptide 105Y(5) .
In this study we examined the
sequence specificity for binding of amyloid- peptide to SEC-R of
the model hepatoma cell line HepG2 and examined the possibility that
SEC-R is expressed on the surface of neurons in primary culture and in
a transformed neuronal cell line.
Since many of these peptides, particularly the amyloid-
peptides, are not soluble in H
0, all were prepared in
Me
SO to ensure that they were completely dissolved and
could be compared with each other.
In
order to determine the distribution of I-amyloid-
peptide-(1-40) during a single cycle of endocytosis we used a
previously described protocol(10, 11) . HepG2 cells or
PC12 cells were incubated for 2 h at 4 °C with
I-amyloid-
-(1-40) in saturating concentrations
(100 nM). Cell monolayers were rinsed and then incubated at 37
°C for several different time intervals in uptake medium alone.
Cell culture fluid was harvested, and cell monolayers were rinsed and
incubated for an additional 60 min in PBS alone or PBS containing
proteinase K as described above to determine proteinase K-resistant
uptake (internal fraction) and proteinase K-sensitive uptake
(surface-bound fraction). Cell culture fluid was counted before (total
extracellular ligand) and after acid precipitation (acid-soluble
extracellular ligand).
Figure 1:
Competition for binding of I-peptide 105Y to HepG2 cells by amyloid-
peptides.
Cells were incubated for 2 h at 4 °C with binding buffer,
I-peptide 105Y in subsaturating concentrations (50
nM) in the absence of competitors (designated 100% total
binding) or in the presence of competitors in several different
concentrations as shown on the horizontal axis. At the end of
this time interval, cells were lysed and cell lysates were subjected to
counting. Results are reported as mean ± 1 standard
deviation for at least three separate determinations. Sequences of the
peptide are compared with the
1-AT sequence at the top. a, peptides 105Y, amyloid-
-(25-35),
amyloid-
-(31-35), and amyloid-
-(1-16). b, peptides amyloid-
-(1-40),
amyloid-
-(1-42), and
amyloid-
-(1-16).
In order to provide further evidence that
amyloid--(25-35) was involved in SEC-R binding and to do
cross-competition studies, we first examined the direct binding of
I-Yam
-(25-35) (YGSNKGAIIGLM) to HepG2 cells (Fig. 2a). There was specific and saturable binding.
Scatchard analysis predicted a k
of
32.1 nM and
430,000 plasma membrane receptors/cell. This is very
similar to the characteristics by which
I-peptide 105Y
binds to HepG2 cells(2) . In separate experiments using
I-amyloid-
-(1-40) and unlabeled peptide 105Y
in HepG2 cells, binding characteristics were also similar to those of
I-peptide 105Y (k
of
35.3
nM and
2.3
10
plasma membrane
receptors/cell). Finally, in cross-competition studies, there was no
significant difference between unlabeled Yam
-(25-35) and
peptide 105Y in competition for binding of
I-Yam
-(25-35) to HepG2 cells (Fig. 2b).
Figure 2:
a, direct binding of I-Yam
-(25-35) to HepG2 cells. Cells were
incubated for 2 h at 4 °C with binding buffer,
I-Yam
-(25-35) in several different
concentrations as shown on the horizontal axis in the absence
of any competitor (Total) or in the presence of unlabeled
Yam
-(25-35) in 200-fold molar excess (Nonspecific).
Cells were then washed extensively and lysed, and cell lysates were
subjected to
counting. Results are reported as mean ± 1
standard deviation for at least three separate determinations.
Scatchard plot analysis is shown in the inset. b,
cross-competition for binding of
I-Yam-
-(25-35) by peptide 105Y. HepG2 cells
were incubated for 2 h at 4 °C with binding buffer,
I-Yam
-(25-35) in subsaturating concentrations
(50 nM) in the absence of competitors (designated 100% total
binding) or in the presence of unlabeled peptide 105Y or unlabeled
Yam
-(25-35) in the concentrations indicated on the horizontal axis. Results are reported as mean ± 1
standard deviation.
Figure 3:
a, direct binding of I-peptide 105Y to PC12 cells. PC12 cells were incubated
for 2 h at 4 °C with PC12 binding buffer,
I-peptide
105Y in several different concentrations as shown on the horizontal
axis in the absence of any competitor (Total) or in the
presence of unlabeled peptide 105Y in 200-fold molar excess
(nonspecific). The difference represents specific binding. Scatchard
plot analysis is shown in the inset. b,
cross-competition for binding of
I-peptide 105Y (left
panel) and binding of
I-Yam
-(25-35) (right panel) to PC12 cells by unlabeled peptide 105Y and
unlabeled amyloid-
-(25-35), exactly as described in the
legend for Fig. 2b. c, direct binding of
I-peptide 105Y to cortical neurons in primary culture.
Exactly as described in the legends for Fig. 3a except
with murine cortical neurons.
Figure 4:
Competition for binding of I-peptide 105Y (a) and
I-Yam
-(25-35) (b) in HepG2 cells by
deleted and substituted amyloid-
peptides. Exactly as described in
the legend to Fig. 1.
Next, we examined several chimeric
amyloid-/
1-AT peptides as competitors for binding of
I-peptide 105Y (Fig. 5a) and
I-Yam
-(25-35) (Fig. 5b)
to HepG2 cells. The amyloid-
/
1-AT chimeric had the
amino-terminal domain of amyloid-
and carboxyl-terminal domain of
1-AT, and the
1-AT/amyloid-
chimera had the reverse.
There were no significant differences in competitive binding efficacy
of these chimeric peptides as compared with each other and as compared
with the original peptides 105Y and amyloid-
-(25-35). In
this design, then, one could not discern a greater role for the
amino-terminal domain of amyloid-
-(25-35) than the
corresponding domain of peptide 105Y in recognition by SEC-R.
Figure 5:
Competition for binding of I-peptide 105Y (a) and
I-Yam
-(25-35) (b) in HepG2 cells by
chimeric amyloid-
/
1-AT peptides. Exactly as described in the
legend to Fig. 1.
Comparison of the amino-terminal region of
amyloid--(25-35) with the corresponding region of peptide
105Y reveals a conserved asparagine-lysine sequence. In peptide 105Y
this is separated from the carboxyl-terminal pentapeptide by a single
proline residue, whereas the sequence in amyloid-
-(25-35) is
separated from the carboxyl-terminal pentapeptide by two residues,
glycine-arginine. We examined the significance of this difference by
generating synthetic peptides in which the intervening proline and
glycine-arginine residues were swapped-i.e. GSNK-P-IIGLM; VKFNK-GA-FVFLM (Fig. 6). The results show no
significant differences from peptide 105Y or
amyloid-
-(25-35) as competitors for binding of
I-peptide 105Y (Fig. 6a) or of
I-Yam
-(25-35) (Fig. 6b). In
contrast, however, substitution of two threonine residues for the two
isoleucine residues at amyloid-
31-32 (peptide
am
-(25-35)TT) markedly decreases the efficacy of competition
for binding to SEC-R. These data provide further evidence that the
carboxyl-terminal pentapeptide is critical for binding to SEC-R but
also suggest that relative hydrophobicity is required even at residues
31-32.
Figure 6:
Competition for binding of I-peptide 105Y (a) and
I-Yam
-(25-35) (b) in HepG2 cells by
swapped amyloid-
/
1-AT peptides. The procedure was exactly as
described in the legend to Fig. 1.
Using the same deleted, substituted, chimeric, and
swapped peptides, the sequence specificity of recognition of
amyloid- peptide by SEC-R was similar, if not identical, in PC12
cells and HepG2 cells (data not shown). Taken together, these data show
that the carboxyl-terminal residues of amyloid-
-(25-35) are
particularly important in binding to SEC-R in hepatoma and neuronal
cells.
Figure 7:
Internalization of I-amyloid-
peptide-(1-40) in HepG2 cells.
Cells were incubated for several different time intervals (horizontal axis) at 37 °C in uptake medium,
I-amyloid-
peptide at saturating concentrations (100
nM), in the absence of competitor (Total), or in the
presence of unlabeled peptide 105Y in 200-fold molar excess (Nonspecific). At the end of each time interval the cells were
washed and incubated for 1 h at 4 °C in phosphate-buffered saline
supplemented with proteinase K. The effect of proteinase K was
terminated by the addition of phenylmethylsulfonyl fluoride (1
mM). Cells were then detached by gentle agitation, pelleted by
centrifugation, and washed, and the pellets were lysed for
counting. Specific internalization represents the difference
between total and nonspecific.
Figure 8:
Distribution of I-amyloid-
peptide-(1-40) in PC12 cells during
a single cycle of endocytosis. PC12 cells were incubated for 2 h at 4
°C with
I-amyloid-
-(1-40) in saturating
concentrations (100 nM), washed extensively, and then
incubated in PC12 medium alone at 37 °C for several different time
intervals as indicated on the horizontal axis. At the end of
each time interval the extracellular medium was harvested, and the
cells were incubated for another 1 h at 4 °C in PBS with proteinase
K. Phenylmethylsulfonyl fluoride was added to 1 mM, and the
cells were pelleted by centrifugation. The supernatant of this initial
centrifugation was counted as the proteinase-K-sensitive cell fraction
(cell surface-bound radioactivity). The cell pellet was washed
extensively in PBS/1 mM phenylmethylsulfonyl fluoride, and
then lysed and counted as the proteinase K-resistant cell fraction
(internalized radioactivity). The extracellular medium was counted
first (total extracellular ligand-dissociated ligand). This medium was
then subjected to trichloroacetic acid-phosphotungstic acid
precipitation, and the supernatant was counted as the acid-soluble
extracellular ligand (ligand degradation products). The results are
representative of three separate
experiments.
The data provide evidence that
amyloid--(1-40), when soluble and recognized by SEC-R, is
internalized and degraded in cells of neuronal origin. Furthermore, the
kinetics of endocytosis and degradation are similar to that for another
SEC-R ligand, the
1-AT-elastase complex (4) .
The results of these experiments show that soluble
amyloid- peptide is recognized by SEC-R on neuronal cells as well
as hepatoma cells. The binding characteristics of SEC-R on neuronal
cells are very similar to those on hepatoma cells. As predicted by
previous sequence comparisons, the SEC-R-binding domain of
amyloid-
peptide lies within amyloid-
-(25-35). This
region is presented to SEC-R to a relatively equivalent extent by
amyloid-
-(1-40), amyloid-
-(1-42),
amyloid-
-(1-39), and amyloid-
-(25-35) itself when
the peptides are prepared and studied in a soluble state. The fact that
soluble amyloid-
-(1-39), amyloid-
-(1-40), and
amyloid-
-(1-42) compete for binding as well as
amyloid-
-(25-35) is important because these peptides have
been found in vivo under physiological
conditions(12, 13, 14, 15, 16) .
There is no evidence that amyloid-
-(25-35), which is much
easier and more economical to use experimentally, is found as a free
peptide in vivo. SEC-R also mediates endocytosis of
amyloid-
peptide in neuronal and hepatoma cell lines. Single
cohort studies show that the internalized amyloid-
peptide is
subjected to intracellular degradation. The kinetics of internalization
and catabolism are very similar to that of another SEC-R ligand, the
1-AT-elastase complex.
Using synthetic peptides based on the
sequences of SEC-R-binding domain of 1-AT and amyloid-
peptide and subjecting them to deletion, substitution, and swapping of
domains, we have described several characteristics of the sequence
specificity by which amyloid-
peptide binds to SEC-R. First, the
carboxyl-terminal pentapeptide (amyloid-
-(31-35), IIGLM)
plays a particularly important role in binding to SEC-R. Substitution
of the carboxyl-terminal methionine with alanine or deletion of two or
three carboxyl-terminal residues results in a marked decrease in
binding. The pentapeptide alone can effectively compete for binding of
I-Yam
-(25-35) and
I-peptide
105Y. Second, the amino-terminal domain of amyloid-
-(25-35)
does not appear to play an important role in SEC-R-binding.
Substitution of alanine for the amino-terminal glycine or deletion of
the two amino-terminal residues did not affect binding at all.
Nevertheless, there was some evidence in the current study which
suggested that the amino-terminal domain of amyloid-
-(25-35)
may contribute to its binding to SEC-R. Peptides in which two or three
carboxyl-terminal residues of amyloid-
-(25-35) were deleted
(amyloid-
-(25-33), Yam
-(22-32)) competed more
effectively for binding of
I-Yam
-(25-35) than
of
I-peptide 105Y. Amyloid-
-(25-33) and
Yam
-(22-32) competed more effectively for binding of
I-Yam
-(25-35) and
I-peptide 105Y
than did a peptide from the corresponding region of
1-AT and of
peptide 105Y, called peptide 105B in previous
publications(3, 4, 5, 6) . Further
evaluation of this issue will require characterization of the structure
of the ligand-binding domain of SEC-R.
There is an asparagine-lysine
sequence within the amino-terminal domain of
amyloid--(25-35) and within the corresponding region of
peptide 105Y, but it is separated from the carboxyl-terminal
pentapeptide by two amino acids, glycine-alanine, in
amyloid-
-(25-35) as compared with one amino acid, proline,
in peptide 105Y. The length or sequence in this region does not appear
to affect receptor binding for either ligand. In contrast, substitution
of the two isoleucine residues at positions 31 and 32 of
amyloid-
-(25-35) with two threonine residues significantly
decreases binding to SEC-R. These data on sequence specificity will be
useful in designing receptor agonists and antagonists.
Taken
together with our previous studies(5, 8) , the data
reported here provide extensive pharmacological evidence that soluble
amyloid- peptide and peptide 105Y, based on the sequence of
1-AT, bind to the same site, called SEC-R, on HepG2 cells, PC12
cells, and neutrophils. First, amyloid-
-(1-42) competes for
cross-linking of an
70-76-kDa radiolabeled polypeptide in
HepG2 cells by a photoreactive derivative of peptide 105Y(8) .
The cross-linking of this
76-kDa polypeptide is highly specific as
shown by positive and negative control competitors and by the absence
of any cross-linking in a SEC-R-negative cell line CHO(8) .
Second, there is cross-competition for binding to HepG2 cells by
I-peptide 105Y and unlabeled amyloid-
-(1-42)
and by
I-amyloid-
-(1-42) and unlabeled peptide
105Y(8) . Third, we show here that the region within
amyloid-
-(1-42) responsible for this cross-competition is
amyloid-
-(25-35) (Fig. 1, a and b)
and that there is cross-competition for binding to HepG2 cells (Fig. 2b) and PC12 cells (Fig. 3b) by
I-peptide 105Y and unlabeled Yam-
-(25-35) and
by
I-Yam-
-(25-35) and unlabeled peptide 105Y.
Fourth, Scatchard plot analysis of binding characteristics of
I-Yam
-(25-35) to HepG2 cells are almost
identical to that of
I-peptide 105Y (Fig. 2a). Fifth, experiments with several series of
peptides based on the sequences of amyloid-
-(25-35) but
modified by deletions, substitutions, or swapping show that these
peptides compete to a similar extent for binding to HepG2 cells of
I-peptide 105Y and
I-Yam-
-(25-35) ( Fig. 4and Fig. 6). Sixth, an
1-AT/am
chimeric peptide, composed
of the amino-terminal domains of peptide 105Y and the carboxyl-terminal
domain of am
, and an am
/
1-AT chimeric peptide, composed
of the amino-terminal domain of am
and the carboxyl-terminal
domain of
1-AT, compete for binding of
I-peptide
105Y and
I-Yam
-(25-35) to HepG2 to a similar
extent (Fig. 5). Seventh, receptor-mediated endocytosis of
I-Yam
-(25-35) is blocked by peptide 105Y (Fig. 6). Eighth, peptide 105Y completely abrogates the
neutrophil chemotactic effect of amyloid-
-(25-35) by
homologous desensitization(3) , providing additional
pharmacological evidence that peptide 105Y and amyloid-
peptide
bind to the same receptor in neutrophils. It will be necessary,
however, to isolate cDNA clones for SEC-R and express SEC-R cDNA in a
heterologous cell to provide definitive evidence that peptide 105Y and
soluble amyloid-
-peptide both bind to it.
The results of this
study may also have important implications for the pathogenesis of
Alzheimer's
disease(17, 18, 19, 20) .
Amyloid- peptide is known to be a major constituent of the amyloid
plaques found in the brains of individuals with Alzheimer's
disease. There is a growing body of evidence that the amyloid-
peptide is generated as a free soluble peptide under physiologic
circumstances as a result of an alternative post-translational
processing
pathway(12, 13, 14, 15, 16) .
In Alzheimer's disease there is greater use of this alternative
processing pathway and more amyloid-
peptide
generated(21, 22, 23) . Presumably, this
amyloid-
peptide accumulates in the brain and over time undergoes
aggregation. A structure termed the ``mature plaque'' is
found in brain during aging. Ultimately there is ingrowth of neurites,
neurofibrillary degeneration, and ingrowth/activation of microglia into
the plaque to form the ``senile plaque'' associated with the
development of
dementia(17, 18, 19, 20) . The
results of the current experiments indicate that SEC-R can mediate
clearance and intracellular catabolism of soluble amyloid-
peptide
in cells that are derived from the central nervous system. Perhaps, it
functions to protect the CNS from excessive extracellular amyloid-
peptide accumulation but gets overwhelmed over time in individuals
predisposed to Alzheimer's disease.
The results of this study
are only applicable to amyloid- peptide that is completely
soluble. In a previous study using radioiodinated amyloid-
peptides there was no evidence for receptor-mediated endocytosis and
degradation in human skin fibroblasts(24) . These results could
be due to the substantial degree of variability in expression of SEC-R
in human skin fibroblast cell lines. Some skin fibroblasts do not
express SEC-R. Even in human skin fibroblast cell lines that express
SEC-R, there is a much lower number of SEC-R molecules/cell than in
HepG2 cells or PC12 cells. (
)It will now be informative to
compare the relative binding of insoluble amyloid-
peptides with
that of soluble amyloid-
peptides, as studied here, in cell lines
such as PC12 and HepG2, which express a substantial number of SEC-R
molecules per cell. A number of studies over the last several years
have shown that amyloid-
peptide-(25-35), and peptides that
contain the region corresponding to amyloid-
-(25-35), when
prepared in an insoluble, aggregated form, are toxic to
neurons(7, 25, 26, 27, 28, 29) .
The most recent of these studies suggest that the toxic interaction of
insoluble amyloid-
peptide-(25-35) with cells is not
sequence-specific but is correlated with formation of insoluble fibrils (30) or correlated with the sequence specificity required for
formation of insoluble fibrils(31) .