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J Biol Chem, Vol. 275, Issue 11, 7935-7941, March 17, 2000
From the GCDFP-15 (gross cystic
disease fluid protein,
15 kDa) is a secretory marker of apocrine differentiation
in breast carcinoma. In human breast cancer cell lines, gene expression
is regulated by hormones, including androgens and prolactin. The
protein is also known under different names in different body fluids
such as gp17 in seminal plasma. GCDFP-15/gp17 is a ligand of CD4 and is
a potent inhibitor of T-cell apoptosis induced by sequential CD4/T-cell
receptor triggering. We now report that GCDFP-15/gp17 is a protease
exhibiting structural properties relating it to the aspartyl proteinase
superfamily. Unexpectedly, GCDFP-15/gp17 appears to be related to the
retroviral members rather than to the known cellular members of this
class. Site-specific mutagenesis of Asp22 (predicted
to be catalytically important for the active site) and pepstatin A
inhibition confirmed that the protein is an aspartic-type protease. We
also show that, among the substrates tested, GCDFP-15/gp17 is specific
for fibronectin. The study of GCDFP-15/gp17-mediated proteolysis may
provide a handle to understand phenomena as diverse as mammary tumor
progression and fertilization.
GCDFP-15 (gross cystic disease
fluid protein, 15 kDa), also known
as prolactin-inducible protein (1), gp17 (2), secretory actin-binding
protein (3), and extraparotid glycoprotein (4), is a protein secreted
by various exocrine glands, including the seminal vesicle, salivary
gland, and sweat glands. This protein is, in addition, expressed by
cancer cells derived from a limited number of tissues, among which are
prominent primary and secondary breast carcinomas exhibiting an
apocrine differentiation (5). The factor exists as a dimer and a
tetramer of a glycosylated 17-kDa subunit in various body fluids (6,
7). GCDFP-15 is a highly specific marker for differential
cytological diagnosis of metastatic mammary tumors (8), and its
expression is up-regulated by androgens, glucocorticoids, and
progesterone (9). However, its role in tumors and the prognostic value
of its expression are not yet clearly established (10). Studies from
our laboratory on the gp17 protein purified from human seminal plasma
have shown that it is a ligand for CD4 (11), in turn a T-cell
co-receptor playing a key role in antigen recognition and T-cell
activation. Further analysis has indicated that early exposure of
peripheral blood T-cells to GCDFP-15/gp17 results in the inhibition of
CD4+ T-cell apoptosis due to CD4 cross-linking and
subsequent T-cell receptor triggering (12). GCDFP-15/gp17 was also
found in the post-acrosomal region of ejaculated spermatozoa and
remains bound to the sperm cell surface after capacitation, thus
implying a possible role in fertilization (13).
In this work, we further defined the properties of GCDFP-15/gp17 with
the aim of gaining a deeper understanding of its role in tumor
progression and reproduction. We found, in fact, that the factor is a
retrovirus-like aspartyl protease and has the potential to modify the
extracellular matrix by fibronectin degradation. The finding that a
significant percentage of breast carcinomas have the ability to
synthesize and secrete GCDFP-15/gp17 (14), together with its absence in
normal resting mammary gland, raises the possibility that this
proteinase might play a role in the lytic processes associated with
invasive breast cancer lesions, as already found for other proteinases,
including matrix metalloproteinases (15), plasminogen activators (16),
and secreted lysosomal enzymes (17).
Gelatin-PAGE1 and
Gelatin/SDS-PAGE Assays--
15% SDS-polyacrylamide and 10%
nondenaturing polyacrylamide gels were prepared, and gelatin (Sigma)
was incorporated at a 0.1% final concentration as originally described
by Heussen and Dowdle (18). Prior to electrophoresis, aliquots of
purified proteins or of proteins from culture supernatants of yeast
were mixed with an equal volume of 2× Laemmli sample buffer with
reducing agent for gelatin/SDS-PAGE and with 0.25 volume of 5×
glycerol-tracking dye buffer (containing 5% glycerol and 0.01%
bromphenol blue) for gelatin-PAGE. After electrophoresis, the
gelatin/SDS-polyacrylamide gels were prewashed with 2.5% (v/v) Triton
X-100 twice for 30 min each to remove SDS and successively as for
gelatin-PAGE were rinsed briefly with buffer (50 mM NaCl
and 25 mM Tris-HCl (pH 7.5)) and incubated in Hanks'
balanced salt solution (Sigma) overnight (16 h) at 37 °C. Following
incubation, the gels were stained with Coomassie Brilliant Blue and
destained according to standard procedures. Protease activity was
revealed by negative staining of transparent bands. The separated
proteins were electrotransferred onto Immobilon-P membrane for 1 h
at 250 mA in a transfer unit (Bio-Rad). The blots were incubated with
specific antibodies (7) and a commercial ECL kit (Amersham Pharmacia
Biotech, Buckinghamshire, United Kingdom). As negative control, the
blots were incubated with the secondary antibody alone, and no reactive
protein was detected.
Modeling Analysis--
The model of GCDFP-15/gp17 was carried
out by the SWISS MODEL server using the ProMod and Gromos 96 programs
for comparative modeling and energy minimization, respectively. The
Swiss Protein Database Viewer program (19) was used for the analysis of
the models. Several structures of the pepsin-like family proteases (1psa, 2psg, 1smr, 5apr, and 1eag) were separately used for modeling, and the best model was obtained with candidapepsin (1eag). The models
were validated with the "What If" program.
Site-specific Mutagenesis--
Site-directed mutagenesis
according to Kunkel (20) was carried out to introduce the desired
mutation (D22S) into the GCDFP-15/gp17 cDNA cloned into plasmid
pET22b(+) (Novagen, Madison, WI) between the EcoRI and
BamHI sites. The oligodeoxynucleotide used in the mutagenesis reaction was 5'-CAGTACGTCCAAATAGCGAAGTCACTGCA-3' (GENSET, Paris, France). The AGC mutant codon in place of the GAC
aspartic acid codon is indicated in boldface. The mutation was verified by DNA sequencing of the complete cDNA. Mutant
GCDFP-15/gp17 cDNA was amplified by polymerase chain reaction to
obtain its 5'- and 3'-end SnaBI and NotI sites to
allow its cloning into plasmid pPIC9. After this cloning, GCDFP-15/gp17
cDNA was checked again by DNA sequencing. The resulting
pPIC9-GCDFP-15/gp17 D22S construct was used to express mutant D22S
protein in Pichia pastoris yeast as described previously
(7).
Proteinase Assays--
Proteinase activity was measured in a
50-µl reaction containing 20 mM NaOAc (pH 3.5), 150 mM NaCl (buffer A), and 20 µg of substrate. Reactions
were initiated by the addition of protease and incubated at 37 °C
for 24 h, and 20 µl of reaction were then analyzed by SDS-PAGE,
followed by Coomassie Blue staining. Human plasma fibronectin was
purchased from Roche Molecular Biochemicals (Mannheim, Germany). The
protein content was determined by the Bio-Rad protein assay using
bovine serum albumin as a standard. The proteins were separated by
SDS-PAGE under reducing conditions according to standard conditions
(6% spacer gel and 10% separation gel). Molecular mass standards
(Bio-Rad) used were myosin (207 kDa),
The GCDFP-15/gp17 protease was incubated in buffer A containing various
inhibitors (purchased from Sigma). Specifically, they were pepstatin A
(1-20 µg/ml), antipain (5 µg/ml), phenylmethylsulfonyl fluoride
(10 mM), aprotinin (10 µg/ml), E-64 (100 µM), and EDTA (10-50 mM). The protease was
first preincubated with each inhibitor at 37 °C for 2 h; the
substrate was then added; and reaction was allowed to proceed as usual.
Structural Homology between GCDFP-15/gp17 and the Aspartyl
Proteinases--
To gain insight into the possible function(s) of
GCDFP-15/gp17, we decided to made full use of the TOPITS
"threading" method (21), a potent means used to search for remote
homologous protein structures with sequence identity around the
so-called "twilight zone" (22), for comparison of the predicted
secondary structure of GCDFP-15/gp17 with those derived from proteins
with known three-dimensional structure present in the Protein Data
Bank, Research Collaboratory for Structural Bioinformatics, Rutgers
University. The highest sequence identity (23% out of an alignment of
141 residues) with a high similarity score was given by an acidic
aspartic proteinase from Candida albicans, also known as
candidapepsin (23). A lower sequence identity (16%) was detected with
pepsinogen (24). Both these enzymes belong to the superfamily of acid
proteinases and, in particular, to the pepsin-like family (25),
suggesting a possible hydrolytic activity of GCDFP-15/gp17. Significant
similarity scores were also observed for a few other hydrolases such as
epidermolytic toxin A from Staphylococcus aureus (1agj) and
neuraminidase from Vibrio cholerae (1kit).
GCDFP-15/gp17 Protease Activity--
We then assessed whether
GCDFP-15/gp17 carries any hydrolytic activity. To do so, the
proteolytic activity of GCDFP-15/gp17, obtained from human seminal
plasma (HSP) of healthy donors by anion-exchange chromatography and
affinity and gel filtration as described previously (7), was examined
by gelatin-PAGE and gelatin/SDS-PAGE. As previously reported (7),
GCDFP-15/gp17 from human seminal plasma exists under these conditions
as a dimeric molecule, which tends to form tetrameric aggregates from
which dimers, however, can be separated. In addition, native monomeric forms can be obtained by a different purification procedure (26). The
dimeric and monomeric forms were thus separately analyzed. Fig.
1A (lane a) shows
that the GCDFP-15/gp17 dimer actually exhibits two electrophoretic
forms (slow (S) and fast (F)) in PAGE
experiments; both forms were reactive in Western blotting with a panel
of anti-GCDFP-15/gp17 monoclonal antibodies, one of which (D6) is shown
in Fig. 1A (lane b). The two bands observed on
PAGE are very likely charge isoforms of GCDFP-15/gp17 exhibiting the
same molecular mass as shown by the following observations. (i) Two
peaks of GCDFP-15/gp17 were resolved by anion-exchange chromatography
(Q-Fast Flow) using a shallower gradient (0-0.3 M) of NaCl
compared with the previously reported procedure (7); (ii) the two peaks
are dimers by gel filtration; and (iii) both are composed of a
different ratio of the four monomeric charge isoforms resolved by
two-dimensional SDS-PAGE (data not shown). The two dimeric forms of the
protein were both endowed with proteolytic activity as shown by a zone of negative staining in gelatin-PAGE in correspondence to the GCDFP-15/gp17 electrophoretic bands (Fig. 1A, lane
c). Furthermore, GCDFP-15/gp17 migrated as a single band under
denaturing SDS-PAGE conditions (Fig. 1B, lane a);
this monomer, after renaturation, was devoid of proteolytic activity
(Fig. 1B, lane b). Similarly, the monomeric form
of GCDFP-15/gp17, purified by the protocol previously described (26),
was devoid of activity in gelatin-PAGE assays (data not shown). In
conclusion, only the dimer of GCDFP-15/gp17 is functionally
relevant.
Aspartate 22 of GCDFP-15/gp17 Is Required for Proteolytic
Activity--
We then generated a multisequence alignment based on the
correspondence of secondary structures (PHDsec method) of GCDFP-15/gp17 to members of known three-dimensional structure of the pepsin-like family (data not shown). The overall sequence identity between GCDFP-15/gp17 and these members of the aspartic proteinase pepsin-like family was low (<25%).
To confirm the threading results and considering both the current lack
of structural information concerning GCDFP-15/gp17 and the absence of a
better structural homolog of known three-dimensional structure, a
homology modeling approach was adopted (Fig.
2A) using, as a reference, the
three-dimensional structure of C. albicans aspartic
proteinase (23) complexed with the A70450 inhibitor (1eag) solved at a
2.1-Å resolution (for details, see Fig. 2A legend). The
structural topological model consisted of three
To further characterize the proteolytic activity of GCDFP-15/gp17, an
expression system in yeast and a purification procedure from
culture supernatants for bona fide recombinant GCDFP-15/gp17 were developed (7). In addition, we exploited the yeast expression system to express a GCDFP-15/gp17 mutant in which Asp22 was
substituted with a serine residue by site-specific mutagenesis. Serine
was chosen since it was not expected to drastically alter the structure
of the catalytic site (29). The basal proteolytic activities of culture
supernatants of a control yeast clone not expressing GCDFP-15/gp17
(GS115/pPIC9), of a yeast clone expressing wild-type recombinant
GCDFP-15/gp17 (GS115/pPIC9-GCDFP-15/gp17), and of a yeast clone
expressing mutant GCDFP-15/gp17 (GS115/pPIC9- GCDFP-15/gp17 D22S)
were thus examined. As shown in Fig.
4A, the wild-type (lane
b) and mutant (lane c) GCDFP-15/gp17 proteins both
exhibited the same electrophoretic pattern of native GCDFP-15/gp17 under nondenaturing conditions, thus showing the presence of two bands
(F and S) that reacted with the panel of
anti-GCDFP-15/gp17 monoclonal antibodies available (data not shown).
The pattern of electrophoretic mobility and immunoreactivity of
GCDFP-15/gp17 D22S confirms that the single amino acid substitution
introduced does not significantly alter the conformation of the
protein. No GCDFP-15/gp17 protein band could be obviously seen in the
supernatant of the GS115/pPIC9 control yeast clone. Analysis by
gelatin-PAGE indicated that although the control supernatant exhibited
only resident yeast proteolytic activity (Fig. 4B,
lane a), the supernatant derived from the clone expressing
wild-type GCDFP-15/gp17 (GS115/pPIC9-GCDFP-15/gp17) also exhibited a
proteolytic activity in the position expected for GCDFP-15/gp17
(lane b). Interestingly, the degrading activity of
GCDFP-15/gp17 was abolished upon introduction of the D22S mutation (lane c, containing a 5-fold excess of proteins from culture
supernatants of the clone expressing GCDFP-15/gp17 D22S).
Fibronectin Is a Specific Substrate of GCDFP-15/gp17--
As
GCDFP-15/gp17 is normally present in human seminal plasma, which
contains proteolytic enzymes implicated in the mechanism of
liquefaction of the seminal coagulum (30), we tested the ability of
GCDFP-15/gp17 obtained from HSP of healthy donors (3) to degrade
fibronectin (31), one of the major components of ejaculated semen
contributing to the formation of the seminal gel (32) and one of the
ligands of GCDFP-15/gp17 (6). Fig. 5A (lanes a and
c) shows that, although fibronectin is known to be highly
susceptible to proteolysis under certain conditions, it is highly
stable in buffer A after incubation for 24 h at 37 °C in the
absence of GCDFP-15/gp17. Conversely, the 220-kDa fibronectin band
(Fig. 5A, lane a) was completely degraded after
incubation with seminal GCDFP-15/gp17 under the same conditions
(lane b). An identical pattern of digestion was observed
when highly purified GCDFP-15/gp17 from breast cyst fluid was used
(data not shown). Furthermore, a homogeneously pure preparation of this
recombinant molecule (r-GCDFP-15/gp17) was tested for the presence of
fibronectin-degrading activity in comparison with the activity of
native GCDFP-15/gp17 from HSP in buffer A. As shown in Fig.
5A (lane d), the pattern of fragmentation of
fibronectin caused by r-GCDFP-15/gp17 was identical to that obtained
when native GCDFP-15/gp17 was employed (lane b).
To determine the substrate specificity of GCDFP-15/gp17, laminin,
vitronectin, or bovine serum albumin in its native form was digested
with the recombinant protease. In all cases, no fragmentation was
observed (see, for example, vitronectin in Fig. 5B).
r-GCDFP-15/gp17 was then tested for its ability to degrade
resorufin-labeled casein, a universal protease substrate used for the
determination of the activity of proteases such as Pronase, trypsin,
endoproteinase Asp-N, and endoproteinase Lys-C. The activity was below
detection (data not shown). Due to the ability of GCDFP-15/gp17 to bind domains 1 and 2 of CD4 (2, 11), we also examined the possibility that
recombinant CD4 can be degraded upon incubation with recombinant GCDFP-15/gp17. As shown in Fig. 5C, also CD4 was resistant
to the proteolytic activity of this factor.
GCDFP-15/gp17 Activity Is Inhibited by Pepstatin A--
The
inhibition of the fibronectin-degrading activity of GCDFP-15/gp17 by
antipain (an inhibitor of trypsin-like serine proteases, papain, and
some cysteine proteases), aprotinin (a broad spectrum serine protease
inhibitor), E-64 (an irreversible inhibitor of cysteine protease),
pepstatin A (an irreversible inhibitor of aspartic proteases such as
cathepsin D, pepsin, and renin), phenylmethylsulfonyl fluoride (an
irreversible inhibitor of serine proteases), or EDTA was then
determined. As predicted by the model and demonstrated by site-specific
mutagenesis, hydrolysis of fibronectin was inhibited only by pepstatin
A, but not by the other inhibitors (Fig. 5D), thus further
supporting the conclusion that GCDFP-15/gp17 belongs to the aspartic
proteinase family.
The gross cystic disease fluid protein GCDFP-15 or
prolactin-inducible protein was independently isolated as an abundant
protein of the fluid of gross cystic disease of the human breast (5) and as a glycoprotein secreted by T47D human breast cancer cells in
response to steroids and lactogenic hormones (9). This protein was also
independently identified as gp17 (2) and secretory actin-binding
protein (3) from human seminal plasma and as extraparotid glycoprotein
(4) from human submandibular/sublingual saliva.
In this report, we provide the first evidence that GCDFP-15/gp17 is a
protease. In particular, we were able to build a model of GCDFP-15/gp17
using, as a guide, candidapepsin (1eag), an aspartic protease from
C. albicans (23); we also observed that GCDFP-15/gp17 displays the "all Reportedly, the aspartic proteases known so far are two-domain proteins
>300 residues in length, with one aspartyl residue in each domain
contributing to the active site (33). By contrast, the GCDFP-15/gp17
sequence does not exceed 118 amino acids in length and contains a
single aspartyl residue at position 22, corresponding to the
Asp32 catalytic residue of domain 1 of the 1eag protease.
The bilobate structure of cellular acidic proteases carrying a two-fold
symmetric axis is believed to have evolved from the duplication of an
ancestral protein whose proteolytically active form was a dimer bearing a similar two-fold symmetry (33). Dimeric retroviral proteases are
considered to be less evolved examples of a progenitor common to
cellular aspartic proteases (25). Duplication and fusion of the cognate
gene would have then allowed divergent evolution, generating a
monomeric protein of ~300 residues with an intrinsic two-fold
symmetry, like the aspartic proteases from lower eukaryotes (23,
33).
We have developed an in vitro assay to check the hydrolytic
activity of GCDFP-15/gp17, and we have demonstrated that it is, in
fact, a protease. By site-specific mutagenesis and by inhibition with
pepstatin A, a specific inhibitor of aspartyl proteinases, we confirmed
that GCDFP-15/gp17 is an aspartic protease. Our results indicate that
only the dimeric form of GCDFP-15/gp17 is active, suggesting that
GCDFP-15/gp17 may function as a retroviral protease. The GCDFP-15/gp17
protease, which is encoded by the prolactin-inducible protein gene onto
the q32-36 region of chromosome 7, may be thus considered either a
living "fossil" of an aspartic protease ancestor or a product of
convergent evolution deriving from mutational changes of the cellular
aspartic protease of the high eukaryotes (33).
As reported above, we found that GCDFP-15/gp17 specifically degrades
the fibronectin molecule under buffer conditions in which the latter
would be otherwise stable. The facts that fibronectin is one of the
major protein constituents of the seminal coagulum (32) and that
GCDFP-15/gp17 constitutes at least 1% of seminal plasma proteins (2)
suggest that GCDFP-15/gp17 may contribute to fibronectin cleavage
during liquefaction.
Furthermore, fibronectin is a multifunctional extracellular matrix
protein that plays a central role in cell adhesion. It interacts in
multiple ways with the cell surface as well as with other extracellular
matrix components and is sensitive to digestion by various proteases
(34, 35). It is possible that fibronectin-degrading proteases, and thus
GCDFP-15/gp17, might facilitate cell invasion by cleaving the
extracellular matrix scaffold between cells, thus detaching cell
membranes from adhesion sites. Consistent with this, a number of
studies have shown a relationship between fibronectin-degrading proteases and cell invasion (36, 37).
In this report, we show the specificity of this protease for the
fibronectin substrate as compared with other extracellular matrix
components such as laminin and vitronectin, which were not degraded by
GCDFP-15/gp17. Thus, GCDFP-15/gp17 produced by tumoral cells may
interfere in matrix deposition. Many studies have reported that the
malignant cells fail to deposit components into the extracellular
matrix in vitro, and many seem to express this defect also
in vivo (38, 39). In particular, fibronectin is among the
matrix components that malignant cells fail to deposit into a matrix.
Finally, given the finding that GCDFP-15/gp17 has been found to block
T-cell apoptosis induced by CD4/T-cell receptor triggering (12) and the
finding that fibronectin-derived peptides modulate apoptotic cell death
(40), it would be interest to assess whether the proteolytic activity
of GCDFP-15/gp17 is required for such an inhibition or whether its
CD4-binding activity is sufficient per se.
We thank C. Vaccaro and V. Carratore for
excellent technical assistance and Dr. E. J. Patriarca for
critical comments.
*
This work was supported by grants from the Associazione
Italiana Ricerca sul Cancro and Consiglio Nazionale delle
Ricerche-Progetto Finalizzato Biotechnology and by Ministero
Università Ricerca Scientifica e Tecnologica Biotechnology
Program L.95/95.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Fax: 39-81-5936123;
E-mail: caputo@iigbna.iigb.na.cnr.it.
The abbreviations used are:
PAGE, polyacrylamide
gel electrophoresis;
HSP, human seminal plasma;
r-GCDFP-15/gp17, recombinant GCDFP-15/gp17.
A Novel Aspartyl Proteinase from Apocrine Epithelia and
Breast Tumors*
§,, and
International Institute of Genetics and
Biophysics and the ¶ Institute of Protein Biochemistry and
Enzymology, Consiglio Nazionale delle Ricerche, via G. Marconi 10, I-80125 Naples, Italy
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (121 kDa),
bovine serum albumin (81 kDa), ovalbumin (51.2 kDa), carbonic anhydrase
(33.6 kDa), and soybean trypsin inhibitor (28.6 kDa). The purity of the
native and recombinant proteins was assessed by silver-stained
SDS-PAGE, PAGE, two-dimensional SDS-PAGE, and NH2-terminal sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A: lane a, 10% nondenaturing
polyacrylamide gel of 1 µg of GCDFP-15/gp17 purified from HSP. The
protein exhibited two forms with different mobility. F (fast
form) indicates the rapidly migrating form, and S (slow
form) indicates the slower migrating form. Lane b,
immunoblot analysis of GCDFP-15/gp17 from HSP against the D6 monoclonal
antibody, a monoclonal antibody raised against GCDFP-15/gp17 from
breast cyst fluid. Electrophoretic conditions were the same as those in
lane a. Lane c, proteolytic activity of
GCDFP-15/gp17 (100 ng) from HSP on a gelatin-polyacrylamide gel.
B: lane a, SDS-PAGE of GCDFP-15/gp17 purified
from HSP (1 µg). Molecular mass standards used were phosphorylase
b (97.4 kDa), bovine serum albumin (66.0 kDa), ovalbumin
(45.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor
(21.5 kDa), and lysozyme (14.4 kDa). Lane b, proteolytic
activity of GCDFP-15/gp17 (100 ng) from HSP on a
gelatin/SDS-polyacrylamide gel.
-sheets shown
schematically in Fig. 2B. The model involves most of the N-terminal domain and only part of the C-terminal domain of 1eag from
Gln11 to Glu202 (Fig. 2). The root mean square
deviation of the 
-carbon atoms was 0.65 Å for a superimposition of
101 residues and 2.08 Å for 115 residues. Asp32 and
Asp218 have been identified as catalytically important for
the active site in 1eag (23). Interestingly, in our model, the
GCDFP-15/gp17 Asp22 is superimposed on 1eag
Asp32. The structural alignment of GCDFP-15/gp17 with some
members of the pepsin-like family is shown in Fig.
3. A search in the Class Architecture
Topology Homologous Superfamily Data Bank (27) indicated that domain 1 of 1eag (chain A) belongs to the class of mainly -proteins with
barrel architecture and topology represented by cathepsin D domain 1 and by several homodimeric retroviral aspartic proteases (28).
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Fig. 2.
Stereo view of GCDFP-15/gp17 superimposed
onto the aspartic proteinase from C. albicans.
A, the GCDFP-15/gp17 molecule is shown as a continuous
thick line; B and C, presented are schematic
diagrams of GCDFP-15/gp17 and 1eag, respectively, showing the secondary
structure elements and the active-site aspartate. The figures were
drawn with WebLab Viewer Version 2.01 (MSI, San Diego, CA).
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Fig. 3.
Structure-based sequence alignment of
GCDFP-15/gp17, candidapepsin (1eag), pepsin (1psa), and submaxillary
mouse renin (1smr). Arrows indicate the -strands
(B). Asterisks indicate residues identical in all
sequences, and dots refer to conservative substitutions. The
letters A-D indicate the -sheets of 1eag. The
lines indicate the -helices (H). The
vertical dotted line separates the aspartic proteinase N-
and C-terminal domains. This alignment was obtained with the Swiss
Protein Database Viewer program and refers only to the superimposed
regions.
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Fig. 4.
A, 10% nondenaturing PAGE of the
proteins (1 µg) from culture supernatants of wild-type GS115/pPIC9
(lane a), recombinant GS115/pPIC9-GCDFP-15/gp17 (lane
b), and mutant recombinant GS115/pPIC9-GCDFP-15/gp17 D22S
(lane c) yeast clones; B, proteolytic activity of
the proteins from culture supernatants (300 ng of proteins for
lanes a and b and 1.5 µg for lane c)
reported in A on a gelatin-polyacrylamide gel. S
and F, slow and fast forms, respectively.
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Fig. 5.
A, proteolytic activity analysis of
GCDFP-15/gp17 from HSP and of r-GCDFP-15/gp17 purified from yeast
against fibronectin in buffer A (25) for 24 h at 37 °C by
SDS-PAGE. Lane a, 8 µg of fibronectin incubated in the
absence of GCDFP-15/gp17; lane b, 8 µg of fibronectin
incubated in the presence of 100 ng of GCDFP-15/gp17; lane
c, 8 µg of fibronectin incubated in the absence of
r-GCDFP-15/gp17; lane d, 8 µg of fibronectin incubated in
the presence of 100 ng of r-GCDFP-15/gp17. Molecular mass standards
used were myosin (207 kDa), -galactosidase (121 kDa), bovine serum
albumin (81 kDa), and ovalbumin (51.2 kDa). B, SDS-PAGE
analysis of vitronectin degradation in the presence (+) or absence (
)
of r-GCDFP-15/gp17. The human purified vitronectin was purchased from
Chemicon International, Inc. (Temecula, CA). Molecular mass standards
used were myosin (205 kDa), -galactosidase (115 kDa), bovine serum
albumin (79.5 kDa), ovalbumin (49.5 kDa), carbonic anhydrase (34.8 kDa), and soybean trypsin inhibitor (28.3 kDa). C, SDS-PAGE
analysis of CD4 degradation in the presence (+) or absence () of
r-GCDFP-15/gp17. Proteolytic activity was determined using 10 µg of
substrate (Genentech, South San Francisco, CA). The same molecular mass
standards used in B were used here. D, SDS-PAGE
analysis of the effect of various inhibitors on GCDFP-15/gp17
proteolytic activity. The same molecular mass standards used in
A were used here. PMSF, phenylmethylsulfonyl
fluoride.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-fold" typical of several aspartic proteinases (25).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Murphy, L. C.,
Tsuyuki, D.,
Myal, Y.,
and Shiu, R. P.
(1987)
J. Biol. Chem.
262,
15236-15241 2.
Autiero, M.,
Abrescia, P.,
and Guardiola, J.
(1991)
Exp. Cell Res.
197,
268-271[CrossRef][Medline]
[Order article via Infotrieve]
3.
Akiyama, K.,
and Kimura, H.
(1990)
Biochim. Biophys. Acta
1040,
206-210[CrossRef][Medline]
[Order article via Infotrieve]
4.
Schenkels, L. C.,
Schaller, J.,
Walgreen-Weterings, E.,
Schadee-Eestermans, I. L.,
Veerman, E. C. I.,
and Nieuw Amerongen, A. V.
(1994)
Biol. Chem. Hoppe-Seyler
375,
609-615[Medline]
[Order article via Infotrieve]
5.
Haagensen, D. E., Jr.,
Dilley, W. G.,
Mazoujian, G.,
and Wells, S. A.
(1990)
Ann. N. Y. Acad. Sci.
586,
161-173[Medline]
[Order article via Infotrieve]
6.
Rathman, W. M.,
Van Zeyl, M. J.,
Van den Keybus, P. A. M.,
Bank, R. A.,
Veerman, E. C. I.,
and Nieuw Amerongen, A. V.
(1989)
J. Biol. Buccale
17,
199-208[Medline]
[Order article via Infotrieve]
7.
Caputo, E.,
Carratore, V.,
Ciullo, M.,
Tiberio, C.,
Mani, J. C.,
Piatier-Tonneau, D.,
and Guardiola, J.
(1999)
Eur. J. Biochem.
265,
664-670[Medline]
[Order article via Infotrieve]
8.
Mazoujian, G.,
Pinkus, G. S.,
Davis, S.,
and Haagensen, D. E., Jr.
(1983)
Am. J. Pathol.
110,
105-112[Abstract]
9.
Shiu, R. P. C.,
and Ivasiow, B. M.
(1985)
J. Biol. Chem.
260,
11307-11313 10.
Hahnel, R.,
and Hahnel, E.
(1996)
Virchows Arch.
429,
365-369[Medline]
[Order article via Infotrieve]
11.
Autiero, M.,
Cammarota, G.,
Friedlein, A.,
Zulauf, M.,
Chiappetta, G.,
Dragone, V.,
and Guardiola, J.
(1995)
Eur. J. Immunol.
25,
1461-1464[Medline]
[Order article via Infotrieve]
12.
Gaubin, M.,
Autiero, M.,
Basmociogullari, S.,
Metiver, D.,
Mishal, Z.,
Culerrier, R.,
Oudin, A.,
Guardiola, J.,
and Piatier-Tonneau, D.
(1999)
J. Immunol.
162,
2631-2638 13.
Bergamo, P.,
Balestrieri, M.,
Cammarota, G.,
Guardiola, J.,
and Abrescia, P.
(1997)
Hum. Immunol.
58,
30-41[CrossRef][Medline]
[Order article via Infotrieve]
14.
Wick, M. R.,
Lillemoe, T. J.,
Copland, G. T.,
Swanson, P. E.,
Manivel, J. C.,
and Kiang, D. T.
(1989)
Hum. Pathol.
20,
281-287[CrossRef][Medline]
[Order article via Infotrieve]
15.
Basset, P.,
Belloq, J. P.,
Wolf, C.,
Stoll, I.,
Hutin, P.,
Limacher, J. M.,
Podhajcer, O. L.,
Chernard, M. P.,
Rio, M. P.,
and Chambon, P.
(1990)
Nature
348,
699-704[CrossRef][Medline]
[Order article via Infotrieve]
16.
Grondahl-Hansen, J.,
Christensen, I. J.,
Rosenquist, C.,
Brunner, N.,
Mouridsen, H. T.,
Dano, K.,
and Blichertoft, M.
(1993)
Cancer Res.
53,
2513-2521 17.
Chauhan, S. S.,
Goldstein, L. J.,
and Gottesman, M. M.
(1991)
Cancer Res.
51,
1478-1481 18.
Heussen, C.,
and Dowdle, E. B.
(1980)
Anal. Biochem.
102,
196-202[CrossRef][Medline]
[Order article via Infotrieve]
19.
Guex, N.,
and Peitsch, M. C.
(1997)
Electrophoresis
18,
2714-2723[CrossRef][Medline]
[Order article via Infotrieve]
20.
Kunkel, T. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
488-492 21.
Rost, B.
(1995)
in
The Third International Conference on Intelligent Systems for Molecular Biology
(Rawlings, C.
, Clark, D.
, Altman, R.
, Hunter, L.
, Lemgauer, T.
, and Wodak, S., eds)
, pp. 314-321, AAAI Press, Menlo Park, CA
22.
Sander, C.,
and Schneider, R.
(1991)
Proteins Struct. Funct. Genet.
9,
56-58[CrossRef][Medline]
[Order article via Infotrieve]
23.
Cutfield, S.,
Marshall, C.,
Moody, P.,
Sullivan, P.,
and Cutfield, J.
(1993)
J. Mol. Biol.
234,
1266-1269[CrossRef][Medline]
[Order article via Infotrieve]
24.
Chen, L.,
Erickson, J. W.,
Rydel, T. J.,
Park, C. H.,
Neidhart, D.,
Luly, J.,
and Abad-Zapatero, C.
(1992)
Acta Crystallogr.
48,
476-488[CrossRef]
25.
Miller, M.,
Jaskolski, M.,
Rao, J. K.,
Leis, J.,
and Wlodawer, A.
(1989)
Nature
337,
576-579[CrossRef][Medline]
[Order article via Infotrieve]
26.
Caputo, E.,
Autiero, M.,
Mani, J. C.,
Basmociogullari, S.,
Piatier-Tonneau, D.,
and Guardiola, J.
(1998)
Int. J. Cancer
78,
76-85[Medline]
[Order article via Infotrieve]
27.
Orengo, C. A.,
Pearl, F. M.,
Bray, J. E.,
Todd, A. E.,
Martin, A. C.,
Lo Conte, L.,
and Thornton, J. M.
(1999)
Nucleic Acids Res.
27,
275-279 28.
Skalka, A. M.
(1989)
Cell
56,
911-913[CrossRef][Medline]
[Order article via Infotrieve]
29.
Grinde, B.,
Cameron, C. E.,
Leis, J.,
Weber, I. T.,
Wlodawer, A.,
Burstein, H.,
and Skalka, A. M.
(1992)
J. Biol. Chem.
267,
9481-9490 30.
Mann, T.,
and Lutwak-Mann, C.
(1981)
Male Reproductive Function and Semen
, Springer-Verlag New York Inc., New York
31.
Ruoslahti, E.
(1988)
Annu. Rev. Biochem.
57,
375-413[CrossRef][Medline]
[Order article via Infotrieve]
32.
Lilja, H.,
Abrahamsson, P. A.,
and Lundwall, A.
(1989)
J. Biol. Chem.
264,
1894-1900 33.
Tang, J.,
James, M. N. G.,
Hsu, I. N.,
Jenkins, J. A.,
and Blundell, T. L.
(1978)
Nature
271,
618-621[CrossRef][Medline]
[Order article via Infotrieve]
34.
Ruoslahti, E.
(1984)
Cancer Metastasis Rev.
3,
45-51
35.
Yamada, K. M.
(1983)
Annu. Rev. Biochem.
52,
761-800[CrossRef][Medline]
[Order article via Infotrieve]
36.
Jones, P. A.,
and Dellerk, Y. A.
(1980)
Cancer Res.
40,
3222-3227 37.
Fairban, S.,
Gilbert, R.,
Ojakian, G.,
Schwimmer, R.,
and Quigley, J. P.
(1985)
J. Cell Biol.
101,
1790-1798 38.
Alitalo, K.,
and Vaheri, A.
(1982)
Adv. Cancer Res.
37,
11-158
39.
Hynes, R. O.,
and Yamada, K. M.
(1982)
J. Cell Biol.
95,
369-377 40.
Fukai, F.,
Mashimo, M.,
Akiyama, K.,
Goto, T.,
Tanuma, S. I.,
and Katayama, T.
(1998)
Exp. Cell Res.
242,
92-99[CrossRef][Medline]
[Order article via Infotrieve]
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