(Received for publication, March 8, 1995)
From the
Angiogenin (ANG) promotes the formation of blood vessels in
animals. This hormone is a small, monomeric protein that is homologous
to bovine pancreatic ribonuclease A (RNase). ANG is a poor ribonuclease
but its ribonucleolytic activity is essential for its angiogenic
activity. RNase is not angiogenic. A hybrid protein was produced in
which 13 residues of a divergent surface loop of ANG were substituted
for the analogous 15 residues of RNase. The value of k
Surface loops tend to be the most divergent regions in
homologous proteins (Kimura, 1982) and appear to play only a minor role
in dictating protein structure (Brunet et al., 1993). Despite
the apparent plasticity of surface loops, their deletion (Kuipers et al., 1989; Pompliano et al., 1990; Baldisseri et al., 1991), insertion (Toma et al., 1991; Wolfson et al., 1991; Braxton and Wells, 1992; Eijsink et
al., 1993; Vuilleumier and Fersht, 1994), or substitution (Harper
and Vallee, 1989; Hynes et al., 1989; Allemann et
al., 1991; Hedstrom et al., 1992) can have a dramatic
effect on protein function. Unfortunately, this effect is usually
deleterious, with any enhanced activities being simple cognates of
those of the wild-type protein.
Angiogenesis refers to the formation
of new blood vessels or neovascularization. In the last decade, a few
proteins that promote angiogenesis have been identified (Folkman and
Klagsbrun, 1987; Folkman and Shing, 1992; Maragoudakis et
al., 1992; Auerbach and Auerbach, 1994; Folkman, 1995). Perhaps
the most intriguing of these proteins is angiogenin (ANG)
The mechanism of
action of ANG is unknown, but the properties of ANG mutants provide
some clues. Amino acid residues have been mutated in the region of ANG
that corresponds to the catalytic active site of RNase. The resulting
proteins are neither ribonucleolytic nor angiogenic (Shapiro et
al., 1989; Shapiro and Vallee, 1989). Thus, the ribonucleolytic
activity of ANG, albeit poor, seems to be essential for its angiogenic
activity. In contrast to active-site mutations, mutations in a
particular surface loop, which is the most divergent part of the two
proteins, eliminate the angiogenic but not the ribonucleolytic activity
of ANG (Harper and Vallee, 1989).
The angiogenic activity of ANG
appears to require two protein sites, one for cleaving RNA and another
for binding to a cellular receptor (Hallahan et al., 1991).
These two functions, both of which are essential for angiogenesis, are
likely to originate from the two parts of the protein highlighted in Fig. 1. We have tested the two-site model of ANG action by
assessing the properties of the surface loop, both when substituted for
the corresponding region in RNase and as an isolated peptide.
Figure 1:
Three-dimensional structure of RNase
(Wlodawer and Sjölin, 1983). The three-dimensional structure of
ANG is similar, except for one surface loop (Acharya et al.,
1994). This divergent surface loop and the conserved catalytic site are
indicated.
Reagents for DNA
synthesis were from Applied Biosystems (Foster City, CA), except for
acetonitrile, which was from Baxter Healthcare (McGaw Park, IL).
Expression vector pET22B(+) and Escherichia coli strain
BL21(DE3) were from Novagen (Madison, WI). Enzymes for the manipulation
of DNA were from Promega (Madison, WI). All other chemicals and
reagents were of commercial reagent grade or better and were used
without further purification.
Ultraviolet
absorbance measurements were made on a Cary model 3 spectrophotometer
(Varian, Palo Alto, CA) equipped with a Cary temperature controller.
Concentrations of UpA were determined by ultraviolet absorption using
The RNase/ANG hybrid protein was produced and
purified as had been other mutants of RNase (Thompson and Raines,
1994; delCardayré and Raines, 1994; 1995; Messmore et
al., 1995; delCardayré et al., 1995). Briefly, E. coli strain BL21(DE3) carrying the mutated plasmid and
induced with isopropyl-1-thio-
The experiments described
herein were based on a hybrid protein in which 13 residues of the
surface loop of ANG (residues 58-70; Fig. 2) were
substituted for the analogous 15-residue sequence of RNase (residues
59-73). Oligonucleotide-mediated site-directed mutagenesis was
used to replace the cDNA that codes for the loop of RNase with DNA that
codes for the loop of ANG. The resulting RNase/ANG hybrid protein was
produced with our existing system for the production of RNase. The
RNase/ANG hybrid protein was >95% pure according to
SDS-polyacrylamide gel electrophoresis as well as zymogram
electrophoresis, which is an extremely sensitive method for detecting
ribonucleolytic activity (data not shown). Gel clot assays showed that
endotoxin contributed <1 ppm to either the purified RNase/ANG hybrid
or the purified RNase A. Three properties of the purified RNase/ANG
hybrid protein were examined: thermal stability, ribonucleolytic
activity, and angiogenic activity.
Figure 2:
Sequence alignment of ANG and RNase based
on structural superposition (Acharya et al., 1994). Residues
with C
Figure 3:
Comparison of cellular infiltration into
discs imbued with a test protein or peptide. Values were obtained by
averaging the penetration for each disc containing a test substance,
which was either a protein (A, 1.0 nmol) or a peptide (B, 10 nmol). The value in B for (58-70)ANG
+ RNase was from discs containing both
substances.
The results of the disc angiogenesis assay of
peptides are shown in Fig. 3B. Discs containing the
(59-73)RNase peptide and (58-70)ANG peptide had 38.1
± 1.9% and 30.1 ± 2.8% penetration, respectively. These
data indicate that (58-70)ANG inhibits angiogenesis, promoting
21% less penetration than did (59-73)RNase. Finally, the results
in Fig. 3B show that (58-70)ANG peptide and RNase
do not work in trans (that is, in an intermolecular manner) to
stimulate angiogenesis.
Angiogenesis has been likened to blood coagulation, in that
it must remain poised but quiescent (Folkman and Klagsbrun, 1987).
Rampant angiogenesis is associated with a wide variety of pathological
conditions. For example, tumor growth and metastasis rely on
angiogenesis, and approximately 50% of a solid tumor consists of blood
vessels and interstitial space (Weinstat-Saslow and Steeg, 1994). In
addition to supplying nutrients to the tumor, these vessels provide a
pathway by which tumor cells enter the circulatory system and
metastasize to remote sites. Angiogenesis also plays a key role in
normal processes that involve tissue growth and development. For
example, the generation of blood vessels is a primary event in the
establishment of the placenta, and is an important feature of the
mammary gland changes associated with lactation (Reynolds et
al., 1992). The healing of wounds and fractures and the success of
limb and organ transplants also require neovascularization.
The goal
of our work is to understand the molecular basis of the angiogenesis
induced by the protein hormone, ANG. We are guided by the two-site
model of ANG action (Hallahan et al., 1991). In this model,
angiogenesis is induced because one site in ANG interacts with a
putative cell-surface receptor, which directs cells to internalize ANG.
The other site catalyzes the cleavage of RNA, perhaps in the nucleolus
(Moroianu and Riordan, 1994). The catalytic site of ANG is conserved
in its homolog, RNase ( Fig. 1and Fig. 2). In contrast,
the surface loop of ANG that may bind to a putative cell-surface
receptor has diverged from that in RNase and all other members of the
RNase superfamily (Beintema, 1987). The sequence of this loop is
conserved, however, in mammalian angiogenins (Bond et al.,
1993).
We have tested the two-site model of ANG action by replacing
the surface loop in RNase (residues 59-73) with the corresponding
region of ANG (residues 58-70). The resulting hybrid protein is
designed to be a molecule that is both an efficient catalyst of RNA
degradation and a strong promoter of neovascularization. Realizing that
mutant proteins like the RNase/ANG hybrid can suffer unintended and
subtle changes in structure or stability (Knowles, 1987), we used
enzymatic activity, which requires the precise alignment of many
nonsequential residues, as a probe of protein structure. The
replacement of residues 59-73 of RNase with residues 58-70
of ANG decreases the specific activity of RNase by only 8-fold (Table 1). This modest effect indicates that the
three-dimensional arrangement of the amino acid residues in the active
site of RNase/ANG is probably similar to that in the active site of
RNase at 25 °C.
The deletion, insertion, or substitution of
surface loops is not likely to have a dramatic effect on the tertiary
structure of a protein (Brunet et al., 1993). Still, such
changes can affect protein stability. The thermal stability of the
RNase/ANG protein was tested, and found to be significantly lower than
that of RNase. Since mice have a body temperature close to the T
The substitution of a surface loop in
RNase with the analogous loop of ANG endows RNase with a distinct, new
activity, the ability to promote angiogenesis (Fig. 3A). Our hybrid protein is the exact complement
of that of Harper and Vallee(1989), who replaced residues 58-70
of ANG with residues 59-73 of RNase. These workers found that
their hybrid protein was unable to induce angiogenesis on the
chorioallantoic membrane of chick embryos. Our strategy is similar to
that of Benner and co-workers, who found that replacing residues
63-74 of RNase with residues 62-71 of ANG resulted in a
60-fold decrease in catalytic activity (Allemann et al.,
1991). Benner and co-workers did not assess the angiogenic activity of
their mutant protein. Our results, along with those of Harper and
Vallee(1989) and Allemann et al.(1991), strongly support the
two-site model of ANG action. In addition, our work provides a rare
example of a protein endowed with a noncognate biological activity
simply by replacing a single element of secondary structure.
The
peptide (58-70)ANG is an inhibitor of endogenous angiogenesis (Fig. 3B). This result is remarkable considering the
inherent conformational entropy of peptides and their susceptibility to
degradation by in vivo proteases. This result portends the
existence of an ANG receptor that can bind to residues 58-70 of
ANG but not to residues 59-73 of RNase. A peptide corresponding
to another region of ANG,(108-123)ANG, has been reported to
decrease significantly the neovascularization elicited by ANG in the
chicken chorioallantoic membrane assay (Rybak et al., 1989).
The similarity of the sequences of (58-70)ANG and
(108-123)ANG (Fig. 4) suggests that these two peptides may
act by binding to the same cellular receptor. Nevertheless, residues
108-123 of ANG are similar to residues 108-123 of RNase (Fig. 2). This region in intact ANG is therefore unlikely to be
involved in receptor binding. Other known peptide/protein inhibitors of in vivo angiogenesis appear to be unrelated to
(58-70)ANG (Ingber and Folkman, 1988; Maione et al.,
1990; Moses et al., 1990; Polakowski et al., 1993).
Figure 4:
Comparison of residues 58-70 and
108-123 of human ANG. Vertical line, identical residues;
colon, similar residues.
The existence in ANG
of a distinct site for each of two functions provides a unique
opportunity to modulate angiogenesis. For example, the results in Fig. 3B suggest that angiogenesis can be inhibited by
interfering with the interaction of ANG and its receptor. Since tumors
require blood vessels to grow, inhibition of blood vessel development
provides a strategy for both preventing the growth of primary tumors
and inhibiting tumor metastasis (Folkman, 1971, 1972; Gimbrone et
al., 1972). Inhibitors of neovascularization would also be useful
in the treatment of rheumatoid arthritis, psoriasis, scleroderma,
hemangiomas, and diabetic retinopathy (Auerbach and Auerbach, 1994).
The results in Fig. 3A suggest that a molecule in
which the ANG surface loop was attached to a ribonuclease (or perhaps,
to another cytotoxin) may be able to promote angiogenesis.
An additional
application of biochemical research on angiogenesis has recently become
apparent. When a coronary artery becomes partially occluded by an
atherosclerotic plaque, the heart muscle normally supplied by that
artery becomes starved for oxygen and other nutrients. In response, the
body grows small collateral arteries to supply viable tissue. In
essence, this response is nature's own version of coronary artery
bypass surgery. Unfortunately, the new collateral arteries are often
unable to compensate for the partial occlusion, and the risk of a heart
attack from the complete occlusion of a main coronary artery remains
high. Unger et al.(1994) have reported that dog cardiac muscle
treated with basic fibroblast growth factor, a protein hormone that
induces angiogenesis, experienced a marked increase in both collateral
artery formation and transmural blood flow. Although these results
portend an exciting treatment for heart-muscle ischemia, protein
hormones are notorious for their side effects. For example, basic
fibroblast growth factor also stimulates the proliferation of cartilage
cells, fibroblasts, and smooth muscle cells. The identification of the
precise regions in protein hormones that are responsible for
neovascularization may allow biological chemists to create molecules
that promote this process, but not others.
We thank Prof. J. F. Riordan for his encouragement, G.
Stephany and Dr. V. R. Muthukkaruppan for invaluable assistance with
the disc and cornea angiogenesis assays, J. E. Thompson for providing
UpA, and Dr. S. B. delCardayré and D. J. Quirk for helpful
advice in various aspects of this research.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/K
for the
cleavage of uridylyl(3`
5`)adenosine by this hybrid protein was
20-fold less than that of RNase but 10
-fold greater than
that of ANG. The thermal stability of the hybrid protein was also less
than that of RNase. Nevertheless, the RNase/ANG hybrid protein promotes
angiogenesis in mice at least as extensively as does authentic ANG.
Thus we present a protein endowed with a noncognate biological activity
simply by replacing a single element of secondary structure. In
addition, a 13-residue peptide corresponding to the surface loop of ANG
inhibits endogenous angiogenesis in mice. These results support a model
in which both a surface loop and a catalytic site are necessary for
the promotion of blood vessel formation by ANG or RNase. The
dissection of structure/function elements in ANG reveals a unique
opportunity to develop new molecules that modulate neovascularization.
(
)(Fett et al., 1985). ANG is a potent
inducer of neovascularization in animals. The human hormone is a
monomeric protein of 123 amino acid residues that is homologous to
bovine pancreatic ribonuclease A (RNase), a paradigmatical enzyme that
catalyzes the degradation of RNA. Although the two proteins are 33%
identical in amino acid sequence, ANG is a poor ribonuclease and RNase
is not angiogenic (Fett et al., 1985).
Materials
Human ANG was from R& Systems (Minneapolis, MN). The
material from R& Systems contains added human serum albumin (HSA)
to stabilize ANG. The same HSA used by R& Systems was obtained
from Miles (Kankakee, IL). Peptides (58-70)ANG and
(59-73)RNase were synthesized by Operon (Alameda, CA). UpA was
synthesized by J. E. Thompson using the methods of Ogilvie et
al.(1978) and Beaucage and Caruthers(1981).
Methods
DNA oligonucleotides were synthesized on an Applied
Biosystems model 392 DNA/RNA synthesizer by using the -cyano
phosphoramidite method (Sinha et al., 1984). DNA sequences
were determined with the Sequenase Version 2.0 kit from U. S.
Biochemical Corp. (Cleveland, OH). Manipulations of DNA were performed
as described by Ausubel et al.(1989).
= 24,600 M
cm
at pH 7.0 (Warshaw and Tinoco, 1966).
Concentrations of protein were determined by ultraviolet absorption
using RNase:
= 0.72 at 277.5
nm, M
13,700; ANG:
= 0.85 at 278 nm, M
14,400;
RNase/ANG:
= 0.70 at 278 nm; M
13,500. The extinction coefficients for ANG and
the RNase/ANG hybrid were calculated from that of RNase (Sela et
al., 1957) with the method of Gill and von Hippel(1989).
Production of RNase/ANG Hybrid Protein
Plasmid pBXR
directs the production of RNase in E. coli (Raines and Rutter,
1989; delCardayré et al., 1995). This plasmid was
constructed by inserting the cDNA that codes for RNase between the MscI and SalI sites in expression vector
pET22B(+). Oligonucleotide-mediated site-directed mutagenesis
(Kunkel et al., 1987) of plasmid pBXR was used to replace the
cDNA that codes for residues 59-73 of RNase with DNA that codes
for residues 58-70 of ANG. The oligonucleotide used was
AGTAGCTCTGTCTCAAGTTTTCTCTGTGTGGGTTACCGTTCTTGTTTTCGCACACGGC, where the
reverse complement of the underlined bases codes for residues
58-70 of ANG.
-Dgalactopyranoside
produced the RNase/ANG hybrid fused to the pelB signal sequence. The
RNase/ANG hybrid protein was solubilized from inclusion bodies,
folded/oxidized in vitro, and purified by fast protein liquid
chromatography on a Mono S column (Pharmacia Biotech Inc.).
SDS-polyacrylamide gel electrophoresis and zymogram electrophoresis of
the pure protein indicated that the pelB signal sequence was removed by
endogenous E. coli proteases, as had been observed with other
mutants of RNase.
Endotoxin Titer
Proteins produced in E. coli can be contaminated with endogenous endotoxins that possess
biological activities. Accordingly, gel clot assays for endotoxin titer
in the preparations of RNase/ANG hybrid and RNase A were performed at
Associates of Cape Cod (Woods Hole, MA). Results were compared to
reference standard endotoxin EC-5 (FDA, CBER) using lot no. 42-134-576
from Pyrotell®.
Thermal Stability
As RNase is denatured, its 6
tyrosine residues become exposed to solvent and its extinction
coefficient at 287 nm decreases significantly (Hermans and Scheraga,
1961). The thermal stabilities of RNase and the RNase/ANG hybrid (which
has 5 tyrosine residues) were assessed by monitoring the change in A with temperature, as described by Pace et
al.(1989). Briefly, the temperature of a solution of protein
(0.1-0.5 mg/mL) in Dulbecco's phosphate-buffered saline
(PBS, which contained (in 1 liter) KCl (0.20 g), KH
PO
(0.20 g), NaCl (8.0 g), and
Na
HPO
7H
O (2.16 g)) was
increased (unfolding) or decreased (folding) in 0.2 °C increments
between 15 and 80 °C, and A
was recorded
after 1 min of equilibration at each new temperature. The data were fit
to a two-state model for denaturation, and used to calculate T
, the midpoint in the thermal unfolding
curve.
Ribonucleolytic Activity
The cleavage of UpA was
monitored by an adenosine deaminase coupled assay (Ipata and Felicioli,
1968) using = -6000 M
cm
. Assays were
performed at 25 °C in O.1 M Mes-HCl buffer, pH 6.0,
containing NaCl (0.1 M), substrate (0.10-0.80
mM), and enzyme (5.5 nM). The values of k
, K
, and k
/K
were
determined from initial velocity data with the program HYPERO
(Cleland, 1979).
Angiogenic Activity
Angiogenesis was assayed using
the disc angiogenesis assay (Fajardo et al., 1988; Polakowski et al., 1993).(
)In the disc assay, a
test substance (1.0 nmol test protein; 10 nmol test peptide) in PBS was
absorbed into the central core excised from a polyvinyl alcohol sponge
disc. The core was then coated with ethylene vinyl copolymer (ELVAX) to
produce a slow-release formulation, and the core was then reinserted
into its polyvinyl sponge disc. The entire disc was then covered on its
two flat surfaces with Whatman No. 1 filter paper made impermeable by
filling the pores with a plexiglass/ethylene dichloride mixture.
Assembled discs were implanted subcutaneously into the flanks of adult
mice (strain BALB/c). Ten discs were implanted for each test substance.
Each mouse carried an experimental disc in one flank and a control disc
(usually RNase) in the other flank. After 10 days in vivo, the
discs were recovered, fixed, and embedded in paraffin. Medial plane
sections (7-10 µm thick) of the discs were prepared and
stained with hematoxylin/eosin to permit visualization of cells that
had penetrated laterally into the disc. The fraction of a particular
section into which cells had penetrated was determined by an image
analysis system using the program OPTIMAS (Jandel Scientific, San
Rafael, CA). All analyses were performed blindly; operators of OPTIMAS
did not know which test substance was on a disc.
Production of RNase/ANG Hybrid
The elaboration
of structure/function relationships in proteins can be facilitated by
using the techniques of recombinant DNA to produce mutant proteins
(Knowles, 1987). Application of these techniques to RNase has been
limited because RNase is notoriously difficult to produce in
heterologous systems, presumably because it is cytotoxic. We have
overcome this barrier by developing an efficient system to produce
RNase in E. coli (delCardayré et al., 1995).
This system allows us to isolate in one chromatographic step enough
pure protein (50 mg/liter of culture) for virtually any type of
biological or biophysical experiment.
positions within 1.2 Å are shown in uppercase letters. The residues of a divergent loop are shown
in a box. Cys-65 and Cys-72, which form a disulfide bond in
native RNase (Fig. 1), are absent from
ANG.
Thermal Stability
The thermal stability of the
hybrid protein was assessed because the protein was to be exposed to
high temperature (38.6 °C) for up to 14 days during in vivo assays for angiogenesis. RNase was found to have T = 66 ± 1 °C in PBS.
In contrast, the hybrid protein was found to have T
= 38 ± 2 °C in PBS. The denaturation of
both proteins was reversible with only minimal hysteresis (data not
shown).
Ribonucleolytic Activity
The ability of the
RNase/ANG hybrid to catalyze the cleavage of RNA was evaluated because
the ribonucleolytic activity of ANG is essential for its angiogenic
activity (Shapiro and Vallee, 1989). As shown in Table 1, the
RNase/ANG hybrid is a potent ribonuclease, having suffered only an
8-fold decrease in the value of k and a 3-fold
increase in the value of K
upon mutation.
The value of k
/K
for the hybrid was 10
-fold greater than that of
authentic ANG.
Angiogenic Activity
The ability of the RNase/ANG
hybrid to promote neovascularization was evaluated by using a disc
angiogenesis assay. In this assay, porous discs were imbued with a
slow-release formulation of a test substance in PBS. The discs were
implanted subcutaneously into mice, recovered after 10 days, and
examined for the penetration of endothelial cells. These results are
shown in Fig. 3. The variation in the penetration of cells into
similar discs implanted into different mice was small, indicating that
mice varied little in their capacity to support angiogenesis.
The
results of the disc angiogenesis assay of proteins are shown in Fig. 3A. Discs containing RNase had 36.4 ± 1.1%
cellular penetration. This value did not differ significantly from that
of discs containing only HSA or PBS (data not shown). In contrast,
discs imbued with the RNase/ANG hybrid protein had 45.3 ± 1.8%
penetration. These data indicate that the RNase/ANG hybrid protein
stimulates angiogenesis in mice, promoting 24% more penetration than
did RNase and 4% more than did authentic ANG. To verify the angiogenic
activity of the RNase/ANG to promote angiogenesis, we also used the
mouse cornea as described (Muthukkaruppan and Auerbach, 1979;
Polakowski et al., 1993). The RNase/ANG hybrid elicited
neovascularization in all six mice tested.(
)
of the hybrid protein, much of the
hybrid protein imbued in a disc may be denatured during in vivo assays of angiogenesis. Only the native form of our hybrid protein
possesses ribonucleolytic and (presumably) angiogenic activity. Thus,
the low thermal stability of the RNase/ANG hybrid protein suggests that
our in vivo assays underestimate the angiogenic potency that
can result from replacement of residues 59-73 of RNase with
residues 58-70 of ANG.
Vallee and co-workers have proposed that actin is the in vivo receptor for ANG (Hu et al., 1993). Actins are abundant
intracellular proteins with an excess of acidic residues (Elzinga et al., 1973). ANG (pI > 9.5) (Fett et al., 1985),
like RNase (pI = 9.3) (Ui, 1971), has an excess of basic
residues (Fig. 2). The affinity of ANG and RNase for actin is
therefore not surprising. Indeed, RNase can compete with ANG for
binding to actin (Hu et al., 1991). Although the affinity of
ANG for actin is quite high (K = 5
10
M) (Hu et al., 1991),
the affinity of ANG for cytosolic ribonuclease inhibitor is much higher (K
= 7
10
M) (Lee et al., 1989). What then is the
significance of the ANG-actin interaction? Recently, we provided
evidence that bovine seminal ribonuclease, a homolog of ANG with potent
cytotoxic activity, has evolved an unusual quaternary structure for the
sole purpose of eluding ribonuclease inhibitor (Kim et al.,
1995). Similarly, a high concentration of actin, as is found in an
endothelial cell, could be effective in shielding ANG (or our RNase/ANG
hybrid) from a low concentration of inhibitor.
(
)(Auerbach and Auerbach, 1994). Thus, understanding
the molecular basis for neovascularization by ANG could lead to a new
class of pharmaceutics that promote blood vessel development. Such
drugs would aid in the treatment of cartilaginous trauma, decubitis
ulcers, wounds, fractures, and transplants.
5`)adenosine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.