(Received for publication, June 29, 1995; and in revised form, August 25, 1995)
From the
Pigment epithelium-derived factor (PEDF), a neurite-promoting
factor, has an amino acid primary structure that is related to members
of the serine protease inhibitor (serpin) family. Controlled
proteolysis of native PEDF (50 kDa) with either trypsin, chymotrypsin,
elastase, or subtilisin yields in each case one major limited product
of 46 kDa as analyzed by SDS-polyacrylamide gel electrophoresis.
N-terminal sequence analysis of the isolated 46-kDa products indicates
a favored cleavage region located toward the C-terminal end of PEDF. A
proteolyzed PEDF protein reaction mixture reveals two overlapping
sequences: that of the N terminus of intact PEDF and that of an
internal region, consistent with cleavage of PEDF about position 382.
These data indicate that PEDF protein has a globular conformation with
one protease-sensitive exposed loop that contains the homologous
serpin-reactive site. Cleavage within the reactive-site loop of PEDF
does not cause a conformational change in the molecules (the stressed (S) relaxed (R) transition) and results in
heat denaturation identical to its native counterpart. This lack of
conformational change is also seen upon cleavage within the
reactive-site loop of the noninhibitory serpin ovalbumin. Furthermore,
the PEDF neurite-promoting function is not lost with cleavage of the
exposed loop. Recombinant PEDF polypeptide fragments with larger
truncations from the C-terminal end show neurotrophic activity. Our
results clearly indicate that integrity of the PEDF homologous serpin
reactive center is dispensable for neurotrophic activity. Thus, the
PEDF induction of neurites must be mediated by a mechanism other than
serine protease inhibition. Altogether our data indicate that PEDF
belongs to the subgroup of noninhibitory serpins and that its
N-terminal region confers a neurite-promoting activity to the protein.
The neurotrophic active site of PEDF is separated from the serpin
reactive-site loop, not only in the primary structure, but also in the
folded protein structure.
PEDF ()was first described as a neurite-promoting
factor that is released by human fetal retinal pigment epithelial (RPE)
cells. It was reported that PEDF isolated from medium conditioned by
human fetal RPE primary cultures promotes neurite outgrowth in cultured
human retinoblastoma Y-79 cells(1) . Information about the PEDF
peptide sequence has permitted the isolation and cloning of a human
PEDF cDNA(2) . From cDNA clones, expression vectors were
constructed (3, 4) and, in turn, specific antisera to
PEDF were developed from the recombinant PEDF
proteins(4, 5) . Antiserum Ab-rPEDF has been
instrumental in the identification of PEDF protein in physiological
sources. PEDF is present in bovine eyes in the interphotoreceptor
matrix (IPM), i.e. the extracellular matrix located between
RPE and the neural retina, and is the sole IPM component responsible
for the IPM neurite-promoting activity(5) . In addition to the
effect on retinoblastoma cells, PEDF has the capacity of promoting
neuronal survival of primary cerebellar granule cell
neurons(6) . These observations support the idea that PEDF is
secreted from the RPE and has a neurotrophic effect on retinal cells.
PEDF is a 50-kDa glycoprotein with a sequence homologous to members
of the serpin family(2, 4, 5) . Sequence
analysis of the 418 amino acids in the human PEDF demonstrates a 27%
identity to -antitrypsin, the serpin prototype.
However, no inhibitory function of PEDF against serine proteases has
yet been demonstrated. Serpins constitute a group of >40 proteins
thought to share the same overall tertiary structure(7) .
Analysis of the serpin folded protein structure in solution indicates
that the C-terminal region of all serpins has an exposed peptide loop
that is highly susceptible to proteolysis. In the case of inhibitory
serpins, the serpin-reactive site, P
, is located within the
exposed loop, and the bond between residues at positions P
and P
` is cleaved by the target protease (where
known). Cleavage of an inhibitory serpin by its target protease induces
a change in conformation from stressed to relaxed (S
R) as revealed by an increase in stability to denaturation.
The residue at position P
binds at the primary specificity
pocket of the target protease, and the protease and serpin form a
complex that impairs further proteolytic activity. It is believed that
the amino acid in position P
helps define the specificity
of inhibition for serpins. Thus, the serpin reactive center is a well
defined structural-functional characteristic of serpin proteins. Not
all serpins have a demonstrable inhibitory activity against a serine
protease, e.g. chicken ovalbumin(8) , rat
angiotensinogen (9) , and barley Z-protein (10) have no
reported inhibitor activities and sequence comparison reveals that they
are also members of the serpin family. As opposed to the inhibitory
serpins, ovalbumin and angiotensinogen do not undergo the serpin
conformational change. The human PEDF sequence has amino acid Leu in
position P
, known to be specific for inhibition of
chymotrypsin and chymotrypsin-like activities; however, the recombinant
PEDF protein (positions 44-418) does not demonstrate inhibition
against these activities(3) . As in ovalbumin, angiotensinogen,
and some dysfunctional serpins, PEDF has residues on the N-terminal
side of the P
residue that are considered unfavorable for
the insertion of the serpin loop into the A
-sheet of the folded
serpin protein. Incorporation of the serpin-exposed loop as an
additional strand into the A
-sheet explains the serpin
conformational change S
R that establishes the
inhibitory status of a serpin.
In this study we have investigated
the overall conformation of PEDF protein and its inhibitory/substrate
status. We have used native PEDF protein purified from bovine eyes (5) and recombinant PEDF polypeptides derived from a human PEDF
expression vector, pRC-BH(3) . Limited proteolysis of native
PEDF with a range of proteases showed that it is consistently
vulnerable to cleavage at sites around the homologous P site (position 382) (
)leaving most of the molecule
resistant to proteolysis. We show that, unlike inhibitory serpins, the
thermal stability of PEDF did not increase upon cleavage at its exposed
loop. PEDF without its exposed loop and even without larger segments
from its C-terminal end retained its neurite outgrowth-inducing
activity. From our results we conclude that PEDF indeed has a folded
protein structure in solution typical of serpins and belongs to the
subgroup of noninhibitory serpins. The N-terminal region of PEDF
contains a neurotrophic active site which is distal from the serpin
reactive loop. The structure-function relationships of the PEDF protein
are discussed.
Recombinant PEDF polypeptides were produced
in Escherichia coli from expression vectors containing human
PEDF cDNA fragments. Preparation of DNA fragments, ligation reactions,
and bacterial transformations were performed as described
before(11) . Deletion mutant plasmids were derived from pRC-BH,
an expression vector with PEDF coding sequences
Asp-Pro
(3) . Plasmid pRC-BH (5.8 kb)
was digested with PvuII and the ends of the 3.2-kb DNA
fragment self-ligated to obtain pRC-BP. Plasmid pRC-BH was digested
with XhoI and HindIII, their ends filled in with
Klenow fragment for a blunt-end ligation to produce pRC-BX (5.2 kb).
Plasmid pRC-BH was digested with Asp718 and HindIII,
their ends filled in with dNTPs and Klenow fragment followed by
blunt-end ligation to obtain pRC-BA (4.8 kb) DNA. PEDF amino acid
positions in the expression plasmids were as follows: BH, 44-418;
BP, 44-267; BX, 44-228; BA, 44-121. Expression of
PEDF genes in bacteria, cell lysis, and protein isolation from
inclusion bodies were as described before for pRC-BH(3) .
Polypeptide fragments BP and BX were further purified by S-Sepharose
chromatography in 4 M urea in 50 mM phosphate buffer,
pH 6.5, and eluted with a NaCl linear gradient. Fractions containing BP
and BX were pooled and stored at -80 °C.
Figure 1: Controlled proteolysis of PEDF with trypsin or chymotrypsin. Trypsin or chymotrypsin was mixed with native PEDF (1.8 µg) at a protease:substrate ratio of 1:100 (w/w). Reaction mixtures were incubated at 25 °C. Proteins were analyzed by 10-20% gradient polyacrylamide-SDS gel electrophoresis under nonreducing conditions. Photograph of a Coomassie Brilliant Blue stained gel is shown. Additions of trypsin (T) or chymotrypsin (CT), and incubation time were as indicated. The arrows on the left denote the positions of substrate and limited product. The numbers on the right correspond to the migration positions of molecular weight standards.
To
determine the position(s) of the cleavage sites, isolated tryptic and
chymotryptic 46-kDa fragments were subjected to automated amino acid
sequencing from their N termini. Both fragments shared the same
sequence with the undigested PEDF protein (Table 1). Sequence
analysis on the intact protein indicated that the N terminus of bovine
PEDF starts at position 23 of the human PEDF precursor
sequence. Note that there is a basic amino acid, Arg, at position 48
that is a potential site for trypsin cleavage, and two aromatic amino
acids, Phe-Phe, at positions 46 and 47 that are potential sites for
-chymotrypsin cleavage. To determine the sequence of the low
molecular weight fragment, analysis was performed on total reaction
mixtures of PEDF treated with trypsin or
-chymotrypsin (at a ratio
of 1:100 for 120 min). Two overlapping sequences were obtained, one
corresponding to the N terminus of intact PEDF starting at Asp
and the other corresponding to the internal C-terminal region
starting at positions 382 and 383 for the tryptic and chymotryptic
fragment, respectively (Table 1). Note that the internal sequence
(20 amino acid residues) shares 95% identity with the human PEDF (see Fig. 7). This sequence has potential sites for chymotrypsin
cleavage, e.g. amino acids Phe (positions 384, 395, and 397),
Leu (positions 386 and 390), and for trypsin cleavage, e.g. Arg (position 399). These results clearly indicate that the 46-kDa
fragments represent the N-terminal domain of the protein and that
limited tryptic and chymotryptic cleavage sites are located between
positions 381/382 and 382/383 of the PEDF molecule. Position 382 maps
to the homologous P
site and is occupied by amino acid Leu.
Figure 7:
Organization of PEDF polypeptide sequence.
The human sequence corresponds to the derived polypeptide from PEDF
cDNA clones(2, 4) . The bovine sequence was determined
by Edman degradation of limited proteolytic fragments of native PEDF (Table 1). Position P is the reactive site in
inhibitory serpins (36) which in the case, Leu
,
corresponds to the position homologous to the P
from the
-antitrypsin sequence. Two distinct regions are
distinguished, a proximal region with a neurotrophic domain located
within positions 44-121, and a distal region, vulnerable to
proteolytic cleavage, that constitutes the homologous reactive-site
loop of serpins, the P
/P
` region. The sequence
of the proximal region has no particular homology with sequences of
other serpins and is apparently unique among them. Sequence analysis of
the proximal region indicates that this region aligns with helices A,
B, C and part of D in the three-dimensional structure of the serpin
prototype(7) . Sequence alignment of the
P
/P
` region of serpins indicates no discernable
sequence conservation; however, the homology is increased at the
flanking regions, the P
- and P
`
regions(3) . A signature pattern developed for the serpin
family by automated assembly of protein blocks for data base (19) is present in the PEDF sequence (positions
388-398).
Proteolytic digestion of PEDF with subtilisin at a
protease:substrate ratio of 1:120 generated a 46-kDa fragment, and
within 60 min of proteolysis, most of the PEDF substrate molecules were
converted to product (Fig. 2, lanes 2 and 3).
Sequence analysis of the isolated 46-kDa fragment indicated that it
started at Thr (Table 1). Treatment with elastase,
at a protease:PEDF ratio of 1:1, also generated a 46-kDa polypeptide
fragment (data not shown), and sequence determination of the 46-kDa
elastase fragment revealed that two-thirds of the molecules began at
Asp
and one-third at Ser
(Table 1). The
loss of nine or seven amino acid residues cannot account for an overall
size reduction of
4 kDa. This implies that PEDF has limited
cleavage sites for subtilisin and elastase located about position 380, i.e. around the P
position. However, treatment of
PEDF with endoproteinase Glu-C or endoproteinase Lys-C, at a higher
protease:substrate ratio (1:24 and 1:57, respectively) than with
subtilisin, did not generate a size reduction of the 50-kDa PEDF
fragment (Fig. 2, lanes 4-7). The N-terminal
sequence of the 50-kDa endoproteinase Glu-C and 50-kDa endoproteinase
Lys-C fragments started at Ala
and at Asp
,
respectively (Table 1). Note that there are several acidic amino
acids, Asp and Glu, at more internal positions, 34, 41, 42, 43, and 44,
that are potential sites for endoproteinase Glu-C cleavage. Thus,
sequence and size analysis of these fragments revealed that the
protease-sensitive region of bovine PEDF does not contain Asp, Glu, or
Lys. The limited tryptic site and the lack of an endoproteinase Lys-C
site suggests that bovine PEDF has Arg around its P
position. In contrast, the known human sequence has Asp and lacks
Arg around P
.
Figure 2: Controlled proteolysis of PEDF protein with subtilisin, endoproteinase Glu-C, or endoproteinase Lys-C. Treatment of native PEDF (0.5 µg) with bacterial enzymes was at the following protease:substrate ratios (w/w): subtilisin, 1:120; endoproteinase Glu-C, 1:24; and endoproteinase Lys-C 1:54. Reaction mixtures were incubated at 25 °C and then analyzed by 12.5% polyacrylamide-SDS gel electrophoresis under reducing conditions. A photograph of a Coomassie Blue-stained gel is shown. Additions of proteases and incubation time were as indicated. Numbers on the left correspond to the electrophoretic migration positions of molecular weight markers.
Figure 3: Effect of PEDF on thrombin. Increasing amounts of native PEDF were mixed with thrombin (0.5 µg) in the absence and presence of heparin (8 units/ml). The reaction mixtures (15 µl) were in 50 mM Tris-Cl, pH 8.3, and 200 mM NaCl, and incubated at 37 °C for 60 min. Proteins were analyzed under nonreducing conditions as described above. Photograph of a 10-20% gradient polyacrylamide-SDS gel is shown. Migration positions of SDS-PAGE standards are indicated to the right of the figure.
Figure 4:
S R transition
assay. Aliquots of intact PEDF and PEDF cleaved by trypsin at its
homologous serpin reactive loop were incubated between 30 °C and
100 °C for 2 h, as described before(13) . Soluble proteins
were then fractionated by centrifugation and analyzed by
SDS-polyacrylamide gel electrophoresis (Panel A). PEDF
proteins were followed by immunoblot against antiserum Ab-rPEDF and
photographs of immunoblots stained with 4-chloro-1-naphthol are shown.
Assay with ovalbumin stained with Coomassie Brilliant Blue is shown as
control. PEDF proteins were quantified by their immunoreactivity
against antiserum to PEDF, Ab-rPEDF (Panel B). Curves in open circles are with intact PEDF and in open triangles with trypsin-treated PEDF.
Figure 5:
Biological activity of PEDF protein
cleaved at its serpin exposed loop by subtilisin. Neurite-outgrowth
assay was as described(3) . Human retinoblastoma Y-79 cells
exponentially growing in serum containing medium were washed twice with
PBS, and plated (2.5 10
cell per ml) in serum-free
minimal essential medium supplemented with a mix of insulin,
transferrin, and selenium. Effectors were then added to the cultures.
After 7 days at 37 °C in 5% CO
, the cells were attached
to poly-D-lysine coated plates with fresh serum-free medium.
The differentiation state of the cultures was monitored at different
intervals after attachment. Morphological characteristics of 9-day
post-attachment cultures are shown. Addition of effectors were as
follows: Panel A, subtilisin-treated PEDF reaction mixture
heated at 75 °C for 20 min added at 100 ng of PEDF protein per ml
of culture; Panel B, reaction mixture as in Panel A minus PEDF.
Figure 6: Biological activity of C-terminal truncated PEDF polypeptide fragments. The neurite-outgrowth assay was as in Fig. 5. Morphological characteristics of 11-day post-attachment cultures are shown. Additions of human recombinant PEDF fragments were as follows: Panel A, BP polypeptide purified from E. coli (pRC-BP), at 50 ng/ml; Panel B, BX polypeptide purified from E. coli (pRC-BX), at 50 ng/ml; Panel C, inclusion bodies extract of E. coli bearing expression plasmid for BA polypeptide (pRC-BA), at 60 ng BA/ml and containing 600 ng/ml total protein; Panel D, inclusion bodies extract of E. coli bearing parent plasmid (pRC23) at 600 ng/ml total protein. Amount of BA polypeptide was estimated from Coomassie Blue-stained gel after SDS-PAGE analysis. Protein samples were in 4 M urea in PBS as described in Becerra et al.(3) .
Analyses of the primary structure of the human PEDF sequence
predict that the PEDF protein shares the common serpin tertiary
structure. By automated assembly of protein blocks for data base
searching (19) the human PEDF protein sequence shows five high
scoring peptide blocks that align with more than 50 serpin sequences
(data not shown). This represents a strong relationship for PEDF to the
serpin family. In addition, its linear sequence (418 residues) contains
small insertions and deletions that are compatible with the
three-dimensional serpin model, i.e. -antitrypsin, and the highly conserved residues
considered critical for maintaining the serpin spatial
structure(7) . However, the actual folded protein conformation
of native PEDF has not yet been investigated. In the studies presented
here, we have used native bovine PEDF protein, which based on its
strong immunoreactivity with antiserum against human PEDF is presumed
to share sequence homology with the human protein(5) . Our data
confirm that the bovine and human sequences are highly homologous
within an internal region (Fig. 7). Controlled proteolysis has
proven successful in analyzing the structural conformation of proteins
in solution and, in particular, the common overall structure of
serpins(20) . Analysis of the PEDF products from several
proteases indicates that the overall native conformation of PEDF
protein includes a highly protease-resistant core consisting of most of
the residues from the N-terminal region, and a protease-sensitive area
located around the homologous serpin-reactive site P
(Table 1). P
is occupied by Leu at position 382
in the bovine and human sequences. Our limited proteolysis data
demonstrate that native PEDF has the common serpin structure, i.e. a globular conformation with an exposed loop located at the
C-terminal end of the molecule. A circular dichroism spectrum confirms
that native bovine PEDF protein contains 35%
structures as shown
for the folded structure of serpins (data not shown). Thus, not only
the linear but also the folded protein conformation of PEDF in solution
is homologous to the serpin family of proteins.
There are now two
serpins that are known to be neurotrophic factors, PN-1/GDN and PEDF.
PN-1/GDN promotes neurite outgrowth from different neuronal cell types,
including neuroblastoma as well as primary hippocampal and sympathetic
neurons(21, 22) , and rescues motor neurons from
naturally occurring and axotomy-induced cell death(23) . The
physiological target for PN-1/GDN has been identified as
thrombin(18, 24) . Sequence comparison between PEDF (2) and PN-1/GDN (25) reveals 23% identity and 48%
homology. ()Human and rat PN-1/GDN have Arg in position
P
, that is specific for thrombin inhibition; while both
human and bovine PEDF have Leu in P
(Fig. 7),
specific for chymotrypsin. However, PEDF (native or recombinant) is not
an inhibitor of
-chymotrypsin
(3) . The effect
of PEDF on thrombin was investigated since HCII, a serpin with Leu in
P
, changes its target specificity to thrombin when in the
presence of sulfated polysaccharides(17) . In contrast to
PN-1/GDN, we have found that PEDF does not decrease the hydrolysis of
thrombin substrate or form serpin:proteinase complexes with thrombin,
even in the presence of sulfated polysaccharides (Fig. 3). Thus,
PEDF is not an inhibitor of thrombin and is structurally and
biochemically distinct from the well established neurotrophic serpin
PN-1/GDN.
To establish the substrate/inhibitor status of PEDF, our
approach was based on the fact that cleavage of inhibitory serpins at
or near the reactive site is followed by a dramatic transformation of
structure from a stressed (S), labile conformation to a more
ordered, heat stable, relaxed (R) form(26) . For
example, cleavage of the reactive center loop of PN-1/GDN,
-antitrypsin and antithrombin III gives the typical
stressed to relaxed (S
R) change in thermal
stability(27, 28) . As opposed to inhibitory serpins,
the noninhibitory serpins do not undergo the S
R conformational change, as detected by heat stability, transverse
urea gels, and even more quantitative
H and
P
NMR spectroscopic data on native and cleaved ovalbumin and
angiotensinogen species(13, 29, 30) .
Comparison of x-ray structures of cleaved forms of
-antitrypsin and ovalbumin supports the conformational
change specific for inhibitory members. The two residues that
constitute the reactive center, P
-P
`, in the
native
-antitrypsin are located 67 Å apart in
the cleaved species while they remain close in the cleaved ovalbumin (31) . However, this lack of S
R transition for noninhibitory serpins has an exception. The hormone
carriers, cortisol- and thyroxine-binding globulins, are reported to
undergo serpin conformational change upon cleavage(32) . It has
been proposed that, in these two noninhibitory serpins, the S
R change is utilized to modulate a different
property of the protein, such as transport in the hormone carrier
globulins. Thermal stability curves for unmodified and cleaved PEDF
reveal that PEDF lacks the S
R conformational
change upon cleavage at its exposed loop (Fig. 4).
Interestingly, PEDF becomes thermodynamically unstable at lower
temperatures than ovalbumin with denaturation temperatures differing by
20 °C. The thermal stability curves for PEDF resemble more the ones
for angiotensinogen than the ones for ovalbumin as reported by Stein et al.(13) and presume that PEDF and angiotensinogen (29) have a greater tendency to denature or unfold than
ovalbumin. The lack of S
R transition for PEDF
may be explained by the presence of unfavorable residues in its
P
region(3) . Recently, Pemberton et al.(33) have reported that a newly identified serpin, maspin,
is not a protease inhibitory serpin. Similar to the study presented
here, the authors show that the tumor suppressor maspin does not
undergo the S
R transition or inhibit
trypsin-like serine proteases. In PEDF, as in angiotensinogen,
ovalbumin, and maspin, the S
R transition may
serve no useful purpose and therefore has been lost by evolutionary
change. Thus, PEDF behaves like a typical noninhibitory serpin.
Our
data demonstrate that while the C-terminal exposed loop is dispensable,
the N-terminal region of PEDF confers a neurotrophic function to the
protein ( Fig. 5and Fig. 6). Consequently, the
neurotrophic induction must be mediated by other than PEDF serpin
inhibition. Experiments with cerebellar granule cells have also shown
that PEDF polypeptide fragments lacking the exposed loop have a
neurotrophic survival effect(6, 34) . From these
observations two distinct regions are identified on the PEDF primary
structure: 1) a proximal region (BA) with a neurotrophic domain located
within residues 44-121 and 2) a distal region with an exposed
loop around position P (Leu
) (Fig. 7).
The sequence of the proximal region is apparently unique, with the
highest degree of homology to members of the serpin family
(20-30%). Spatial models for PEDF based on the three-dimensional
structures of
-antitrypsin and ovalbumin (7, 35) show that the proximal BA region, composed of
putative
-helices A, B, C, and part of D, is located to the
opposite pole from the exposed loop. This model is in agreement with
the fact that the PEDF neurotrophic activity is not lost with cleavage
of the exposed loop. Thus, in the folded protein structure of PEDF the
neurotrophic domain is also separated from the exposed loop.
Altogether, the results presented here demonstrate that PEDF has the protein conformation of a serpin, but inhibition of serine proteases by PEDF cannot account for its neurotrophic activity. The initial biochemical step(s) for the complex biological effect of neurite outgrowth is still unknown. However, the relationships between structure of PEDF and its biological function suggest that the mode of action would include the interaction(s) between peptide residues from its proximal BA region and molecules associated to the membrane of target cells that would then trigger subsequent events of signal transduction.