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J Biol Chem, Vol. 274, Issue 41, 29352-29357, October 8, 1999
From the Fibroblast growth factor (FGF)-9 is a
glycosylated neurotrophic polypeptide highly expressed in brain. The
mechanism for its secretion from expressing cells is unclear, because
its primary structure lacks a cleavable signal sequence. We, therefore,
investigated the mechanism and structural requirements for secretion of
FGF-9. As with other secreted proteins, in vitro
translation of FGF-9 was inhibited by signal recognition particle,
which binds to the signal sequence. When translated in
vitro, full-length FGF-9 was translocated into microsomes,
glycosylated, and protected from trypsin digestion. By using various
FGF-9 deletion mutants, we found that two hydrophobic domains, located
at the N terminus and at the center of the FGF-9 primary structure,
were crucial for translocation. Examination of various point mutants
revealed that local hydrophobicity of the central hydrophobic domain,
but not the N terminus, was crucial for translocation. Analogous
results were obtained with respect to FGF-9 secretion from transfectant cells. Upon deletion of the complete sequence preceding it, the previously uncleavable hydrophobic domain appeared to serve as a
cleavable signal sequence. Our results suggest that nascent FGF-9
polypeptides translocate into endoplasmic reticulum without peptide
cleavage via a co-translational pathway in which both the N
terminus and the central hydrophobic domain are important; thereafter,
FGF-9 is glycosylated and secreted.
Fibroblast growth factors
(FGFs)1 constitute a family
of related polypeptides that are involved in a variety of biological processes including morphogenesis, angiogenesis, and tissue remodeling (1-6). FGFs stimulate proliferation of a wide range of cell types including those of neuroectodermal origin (7-9). In the presence of
heparin/heparan sulfate proteoglycans, FGFs bind with varying affinities to transmembrane tyrosine kinase receptors encoded by four
distinct genes (10). The binding induces receptor dimerization, activation of the intracellular kinase domain, and downstream intracellular signaling (11). In this context, we sought to understand
better the mechanism by which FGFs are secreted from expressing cells.
FGF-9 was originally purified as a glial cell-activating factor from
culture medium conditioned with the human NMCG-1 glioma cell line.
Indeed, FGF-9 is a potent mitogen for glia, rat primary cortical
astrocytes, BALB/c3T3 fibroblasts, and oligodendrocyte type 2 astrocyte
progenitor cells (12). Despite the absence of a typical N-terminal
signal sequence, FGF-9 is efficiently secreted following transfection
into COS cells (13). Peptide microsequencing revealed that secreted
FGF-9 has an alanine as the second amino acid residue at its N terminus
(14); it is N-glycosylated following secretion or in
vitro translation in the presence of microsomes, which is in good
agreement with the presence of an N-glycosylation consensus
sequence (13, 15); and deletion of the N-terminal 33 amino acids of
FGF-9 abolished the secretion. These characteristics distinguish FGF-9
from FGF-1 and FGF-2, both of which lack a secretion signal and are
only inefficiently exported from cells in non-glycosylated forms. In the present study, we investigated the mechanism and structural requirements for the secretion of FGF-9.
Reagents and Cell Culture--
Wheat germ extract and rabbit
reticulocyte lysate were purchased from Promega Corp. (Madison, WI).
COS-1 cells were cultured with Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum. All restriction
enzymes were purchased from Takara Syuzo (Kyoto, Japan).
Construction of Plasmids--
Various deletion mutants of FGF-9
were generated by PCR with the primers listed in Table
I using FGF-9 cDNA in the pUC118 vector (R-6) as a template. The mutants were cloned into a
pBluescriptIIKS(+) expression vector (Stratagene La Jolla, CA) as
described previously (16). Briefly, a representative PCR reaction
mixture would consist of Vent DNA polymerase (New England Biolabs,
Beverly, MA), 20 µM dNTPs, 1 µg of primer (Table I), 1 µg of template DNA (full-length FGF-9) and 1 unit of enzyme in a
final volume of 50 µl. The amplification protocol consisted of 30 cycles at 94 °C for 1 min, 72 °C for 2 min, and 50 °C for 2 min (16). The amplified product of each construct was separated by
agarose gel electrophoresis, and a fragment with the predicted size was
recovered from the gel using Geneclean (Bio 101, Inc., Vista, CA). The
fragment was then digested with EcoRI and BamHI
and then subcloned into the EcoRI/BamHI site of
the pBluescriptIIKS(+) vector. Nucleotide sequences of all constructs
were confirmed by DNA sequencing (Applied Biosystems model 373A).
The desired point mutants were constructed using the overlap extension
method (17) with the primers listed in
Table II; they include
Leu25-Asn, Gly71-Tyr, Gly80-Tyr,
Ile98-Asn, Ile100-Asn, and
Ala101-Asn. Briefly, PCR was first carried out with primers
I and II and then with primers III and IV. Primer I corresponds to the 5'-end of FGF-9; primers II and III contain the sense and antisense strands of the mutation site; and primer IV corresponds to the complementary 3'-end of FGF-9. The first and the second PCR products were purified by agarose gel electrophoresis and Geneclean (Bio 101).
Both products were then mixed and subjected to PCR using primers I and
IV. Each PCR reaction mixture consisted of AmpliTaq DNA polymerase
(Perkin-Elmer Applied Biosystems, Forster City, CA), and the
corresponding buffer was provided by the manufacturer (20 µM dNTPs, 1 µg of each primer (Table II), and 1 unit of
enzyme) in a final volume of 50 µl. The amplification protocol
consisted of 30 cycles at 94 °C for 1 min, 72 °C for 2 min, and
50 °C for 2 min (16). The amplified product was separated and
recovered and then subcloned into a pGEM-T vector (Promega Corp.). The
cDNA cloned into the pGEM-T vector was digested with
EcoRI and BamHI and subcloned into the
EcoRI/BamHI site of the pBluescriptIIKS(+) vector. All nucleotide sequences were confirmed by DNA sequencing (Applied Biosystems model 373A).
For expression of FGF-9 in COS-1 cells, the various FGF-9 cDNAs in
pBluescriptIIKS(+) were digested with EcoRI and
XbaI and subcloned between the EcoRI and
XbaI sites of a pSVK3 vector (Amersham Pharmacia Biotech).
The expression vector for FGF-9 N33(34-208) was previously constructed
(14).
In Vitro Translation of FGF-9 and Its Arrest by Signal
Recognition Particle (SRP)--
Purified SRP as well as plasmids
containing preprolactin (PPL) or mature regions of preprolactin
( In Vitro Translation and Microsomal Translocation of FGF-9 Using
the Reticulocyte Lysate System--
The plasmids constructed for the
in vitro translation assay were linearized by digestion with
BamHI, and each cDNA was transcribed using the mMESSAGE
mMACHINE T3 in vitro transcription kit (Ambion Inc.). The
transcript was then translated using nuclease-treated rabbit
reticulocyte lysate (Promega Corp.) at final concentration of 33%,
with or without canine pancreatic rough microsomes prepared as
described previously (18). Translation was performed at 30 °C for 30 min, and [3H]leucine was included in the reaction
mixture. In some cases, aliquots of the translation products were
further treated with 0.25 mg/ml trypsin (Sigma) for 1 h, on ice,
in the presence or absence of 0.6% Triton X-100 (Nacalai Tesque,
Tokyo, Japan). The translation products of the deletion mutants were
precipitated by adding (NH4)2SO4 to
65% saturation, washed with 5% trichloroacetic acid, and dissolved in
SDS-PAGE sample buffer by sonication. They were then resolved on
SDS-polyacrylamide gels and fixed. The gels were soaked in ENLIGHTENING
(NEN Life Science Products) for fluorography, dried, and the
translation products were visualized by Biomax MS film (Kodak).
Analysis of FGF-9 Expressed by COS-1 Transfectants--
COS-1
cells were transfected using the DEAE-dextran method. Cells were plated
on 10-cm dishes and cultured until 40% confluent. They were then
washed with phosphate-buffered saline (PBS) and incubated in 3.5 ml of
DNA mixture (20 µg of DNA in 0.4 mg DEAE-dextran/ml of
Tris-supplemented, serum-free medium (Tris-SFM, 50 mM
Tris-HCl, pH 7.5) in DMEM) for 4 h at 37 °C; after that the
cells were washed again in PBS and placed in 7 ml of 100 µM chloroquine in Tris-SFM. After incubating for 4 h
at 37 °C, the cells were once again washed in PBS and further
cultured for 3 days in 4 ml of DMEM supplemented with 10% fetal bovine
serum. The conditioned medium was collected, centrifuged at 10,000 × g for 10 min at 4 °C, and then passed through 0.2-µm
Millex-GV filters (Millipore Bedford, MA). Fifty µl of
heparin-Sepharose beads (Amersham Pharmacia Biotech) were added to the
filtrate, incubated for 3 h at 4 °C, and recovered by
centrifugation. The proteins adsorbed on the beads were recovered by
boiling them in SDS-PAGE sample buffer.
Treatment with N-glycosidase was carried out according to
the manufacturer's protocol. Samples were incubated for 16 h at 37 °C with 0.4 units of N-glycosidase F (Roche Molecular
Biochemicals) in a solution containing 5 mM Tris-HCl (pH
8.0), 0.2% SDS, 0.5% Nonidet P-40, 1% glycerol, and 1%
2-mercaptoethanol. To prepare cell lysates, the cells were washed with
PBS and treated with 10% trichloroacetic acid for 30 min on ice. The
cells were then collected and dissolved in SDS-PAGE sample buffer by
sonication. The proteins were resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and blotted with anti-FGF-9 antibody (13).
The resultant signals were detected using horseradish
peroxidase-conjugated anti-rabbit IgG and ECL (Amersham Pharmacia Biotech).
FGF-9 Lacks a Predictable Signal Sequence but Is Secreted as an
N-Glycosylated Form--
We analyzed the primary structure of FGF-9
for the presence of a signal sequence using the latest prediction
software available on the web server (SignalP (19)). Consistent with
previous experimental results, we found that FGF-9 lacked a predicted
signal sequence.
To confirm the secretion of FGF-9, COS cells were transfected with an
expression vector encoding the full-length FGF-9 cDNA. Subsequently, cell lysate and conditioned medium were then analyzed for
FGF-9 content (Fig. 1). The conditioned
medium (lane 2) contained high levels of FGF-9, as compared
with cell lysates (lane 1); secretion was apparently quite
efficient. Two forms of FGF-9 with molecular masses of 30 and 27 kDa
were present in the conditioned medium (lane 2, closed and
open arrowheads, respectively). Levels of 30-kDa FGF-9 were
diminished by treating the conditioned medium with
N-glycanase (lane 3, open arrowhead), which
suggests that the 30-kDa moiety is the N-glycosylated form
of the 27-kDa FGF-9 simple polypeptide and is consistent with the
presence of an N-glycosylation consensus sequence within its
primary structure
(Asn79-Gly80-Thr81).
SRP Inhibits in Vitro Translation of FGF-9--
The transport of
proteins containing signal sequences across the endoplasmic reticulum
(ER) membrane in mammalian cells generally occurs in a co-translational
manner. The process begins in the cytosol with a targeting phase; the
signal sequence of a nascent polypeptide chain that has emerged from
the ribosome is recognized by the 54-kDa subunit of SRP (20, 21), which
in turn targets the protein to the ER membrane. Once SRP binds to the
signal sequences of a nascent chain, it halts or slows elongation of
the polypeptide until contact is made with a docking protein (SRP
receptor) in the membrane (22, 23). Consequently, in the absence of
membrane, SRP strongly suppresses translation of proteins with signal
sequences. To examine the involvement of SRP in FGF-9 synthesis, we
used a wheat germ, in vitro translation system in which
neither SRP nor microsomes were present. As shown in Fig.
2, addition of SRP strongly inhibited
translation of preprolactin (PPL), a well characterized, secreted
protein containing a signal sequence. In contrast, translation of
Both the N Terminus and the Central Hydrophobic Region of the FGF-9
Primary Structure Are Indispensable for Translocation across Microsomal
Membranes--
To determine the sequence in FGF-9 responsible for its
secretion, we conducted a microsomal translocation assay using a rabbit reticulocyte lysate, in vitro translation system containing
endogenous SRP and other components. As shown in Fig.
3A, FGF-9 was translated as a
27-kDa polypeptide (open arrowhead), which is in good
agreement with the calculated molecular mass of the protein
(lanes 1 and 2). When microsomes were added to
the translation reaction, an additional 30-kDa translation product
resulting from N-glycan modification at the Asn-Gly-Thr
sequence was observed (Fig. 3A, lanes 3 and 4, closed
arrowhead, and Fig. 3G). With the exception of the
30-kDa product (lane 7), all of the translation products were digested upon addition of trypsin to the reaction mixture (lanes 5-8). Furthermore, pretreatment with Triton X-100
prior to trypsinization resulted in complete degradation of the
translate, including the 30-kDa product (lane 8). This
confirmed that the 30-kDa protein in lane 7 was a
glycosylated form of FGF-9 that was protected from trypsin digestion by
translocation into the microsomes.
By using the same protocol with five deletion mutants, we then
identified the structural requirements for FGF-9 translocation (Fig.
3G). The dFGF-9-(1-104) mutant, which lacked the C-terminal half of the molecule, was translated as a 11-kDa polypeptide (Fig. 3B, lane 1, open arrowhead). This translation product was
not protected from trypsinization (Fig. 3B, lane 7),
indicating that it was not translocated into microsomes. This
conclusion was confirmed by the absence of a glycosylated form of the
polypeptide (Fig. 3B, lanes 3 and 4), even though
the N-glycosylation sequence was present. Both
dFGF-9-(105-208) and dFGF-9-(53-156) were also not translocated into
microsomes (Fig. 3, C and D). On the other hand, dFGF-9-(1-156) was both glycosylated (Fig. 3E, lanes 3, 4, and 7) and protected from trypsinization
(lane 7) in a manner similar to full-length FGF-9. Thus,
both the N terminus and the central hydrophobic region of the FGF-9
primary structure appear to be indispensable to translocation, whereas
the C-terminal one-fourth of the full-length FGF-9 is apparently not required.
Interestingly, deletion of amino acid residues 1-90 preceding the
central hydrophobic domain yielded a polypeptide that was not
glycosylated yet was capable of being translocated (Fig.
3F). Although the dFGF-9-(91-208) translation product has a
molecular mass of 13 kDa (lanes 1 and 2, open
arrowhead), in the presence of microsomes, another molecular
moiety of 11-kDa was produced (lane 3, arrow). The 11-kDa
protein, but not the 13-kDa one, was protected from trypsinization
(lane 7) and, therefore, must have been translocated.
Because dFGF-9-(105-208), which is identical to dFGF-9-(91-208)
except the absence of peptide (91-104), is not translocated nor
processed, dFGF-9-(91-208) is most likely cleaved at its N terminus.
Analysis of the primary structure of dFGF-9-(91-208) using SignalP
software revealed a likely signal peptide and predicted a cleavage site
between positions 112 and 113 (Val-Asp-Ser Local Hydrophobicity of the Central Hydrophobic Domain, but Not of
the N Terminus, Is Important for FGF-9 Translocation--
A hydropathy
plot of FGF-9 showed that two major hydrophobic regions are contained
within its primary structure as follows: a weak hydrophobic domain at
the N terminus and a strong hydrophobic domain at the center of the
polypeptide (Fig. 4C). Because
the aforementioned results suggested the involvement of SRP in the secretion of FGF-9 (Fig. 2), we constructed various FGF-9 point mutants
in which local hydrophobicity was significantly altered by amino acid
substitution (Fig. 4, A and C). Substituting Asn for Leu25 makes the N terminus relatively less hydrophobic
(Fig. 4C). Unexpectedly, FGF-9(L25N) was protected from
trypsinization, indicating translocation of the mutant into microsomes
(Fig. 4B); in contrast, FGF-9(I98N) and FGF-9(I100N, A101N)
were not protected (Fig. 4B). Again, the protected bands
were observed as the glycosylated forms of the molecules (lane
7 of wt FGF-9, FGF-9(L25N)), which means that the
translocated proteins were fully accessible to the glycosylation machinery. To ascertain whether a structural interaction between the N
terminus and the central hydrophobic domain affected translocation, point mutations (FGF-9(G71Y) and FGF-9(G80Y)) were introduced that
caused decreased flexibility of the polypeptide. Despite the reduced
flexibility, these mutants were also protected from trypsinization
(Fig. 4B).
FGF-9 Mutants Capable of Microsomal Translocation Are Processed in
and Secreted from COS Transfectants--
After confirming that FGF-9
and FGF-9(L25N) were translocated into microsomes and that FGF-9(I100N,
A101N) was not, secretion of these mutants from COS transfectants was
examined. We found that secretion from COS cells corresponded with
in vitro microsomal translocation. When culture supernatants
of the COS transfectants were adsorbed onto heparin-Sepharose beads and
resolved by SDS-PAGE, glycosylated forms of FGF-9 and FGF-9(L25N) were
clearly detected (Fig. 5, lanes
5 and 6, closed arrowhead). FGF-9(I100N, A101N), by
contrast, was not detected in the supernatant (lane 7),
although its simple protein was translated in the cells (lane 3, open arrowhead). We also confirmed the previous observation that
FGF-9N33-(34-208) (Fig. 5, lane 11, open arrowhead) was
neither secreted nor glycosylated (Fig. 5, lanes 11 and
14). In addition, the dFGF-9-(91-208) expressed in COS
transfectants was indeed detected at 13 and 11 kDa (Fig. 5, lane
15, arrowhead and arrow, respectively), the same sizes as those observed in in vitro translation/translocation
experiment (Fig. 3F, lane 3). By in vitro
translation without inclusion of microsomes, only the unprocessed
13-kDa form was detected (Fig. 5, lane 16, arrowhead).
In the present study we showed that 1) SRP arrests translation of
FGF-9; 2) FGF-9 is translocated into microsomes where it is
glycosylated; 3) both the N terminus and central hydrophobic regions
are important for translocation; 4) the hydrophobicity of the central
hydrophobic region, but not that of the N terminus, is crucial for
translocation; 5) the central hydrophobic region appears to serve as a
cleavable, N-terminal signal sequence in an artificial construct; and
6) point mutations that abolished microsomal translocation of FGF-9
also abolished its secretion from cells. These results indicate that
despite the absence of a predictable, cleavable signal sequence,
secretion of FGF-9 from expressing cells is mediated via
co-translational translocation into ER in a manner very similar to that
of other secreted proteins.
Proteins secreted by co-translational transport generally contain
N-terminal signal sequences. When translated on rough ER, nascent
proteins to be secreted are targeted to translocation channels located
in the ER membrane, and the signal sequence is cleaved by a signal
peptidase (24). The specific structures of signal sequences are
diverse; nonetheless, they have certain features in common as follows:
1) a net positive charge at the N terminus, 2) a hydrophobic region,
and 3) a C-terminal region with small, nonpolar amino acids at
positions Our findings that the C-terminal hydrophilic region (i.e.
amino acids 157-209) was not required for FGF-9 translocation (Fig. 3E) and that translation of FGF-9 was inhibited by SRP,
which generally binds hydrophobic motifs in signal sequences, prompted us to characterize the contribution made by the FGF-9 hydrophobic regions to its translocation into microsomes. FGF-9 deletion mutants that lacked any part of the major hydrophobic regions were not translocated (Fig. 3G). Moreover, as substituting Asn for
Ile98 or Asn-Asn for Ile100-Ala101
blocked translocation, local hydropathy of the central hydrophobic domain is apparently crucial for translocation. Analogous observations were made with plasminogen activator inhibitor-2 and ovalbumin, SERPIN
family proteins which are the only other known examples of secreted
proteins lacking cleavable signal sequences (26-29). With respect to
plasminogen activator inhibitor-2, it was shown that SRP recognizes the
second hydrophobic region in its primary structure and that this region
was important for translocation into the ER (30). Similarly, the second
hydrophobic region was also necessary for efficient secretion of
ovalbumin (28). In contrast, despite the finding of a previous study
(14) that the N-terminal 33 amino acids were indispensable for FGF-9
secretion, making this region less hydrophobic by substituting Asn for
Leu25, did not affect translocation. Thus, whereas both the
N terminus and central hydrophobic region (amino acids 95-120) were
required for translocation, the specific requirement for hydrophobicity was only demonstrated for the central region.
Point mutations (G71Y and G80Y) that should have restricted local
flexibility of the polypeptide did not affect translocation. It is
unlikely, therefore, that an interaction between the N terminus and the
central hydrophobic domain is important for translocation. Consequently, it is still unclear how the N terminus of FGF-9 is
involved in the secretion mechanism. Our findings that the central
hydrophobic domain is able to function as a signal sequence, but that
the presence of excess amino acids preceding this region (amino acids
34-90) abolished the secretion, suggest that the N terminus is
involved in the effective presentation of the central hydrophobic
region to the translocation machinery. Similar observations have been
made with plasminogen activator inhibitor-2, where a weakly hydrophobic
region preceding a more strongly hydrophobic one is important for the
translocation of the protein into microsomes (31).
Finally, we have identified FGF-9 as the first example, outside the
SERPIN family, of a secreted protein that translocates into microsomes
with a clearly uncleavable signal sequence. We have also shown that a
sequence of amino acids within the central hydrophobic domain of FGF-9
appears to serve as a cleavable signal sequence when placed at the N
terminus. The molecular mechanism by which an otherwise cleavable
signal sequence can serve as an uncleavable signal sequence when it is
located in the middle of a polypeptide awaits further investigation. A
similar secretion mechanism may be shared by other recently identified
members of the FGF family that also lack signal sequences (32), for
instance FGF-16, whose hydropathy plot reveals a central region very
similar to that of FGF-9. The present results are consistent with the conclusion that FGF-9 is secreted via a co-translational pathway. However, because the translocation machinery and the steps in this
process are not fully characterized, the possibility that FGF-9 is
secreted by an alternative process (e.g. post-translational) cannot be fully excluded.
We thank Prof. Kazunari Taira at Tsukuba
University for support and Dr. Masashi Suzuki at NIBH for technical
advice. The text was edited by Dr. William Goldman at MST Editing Co.
*
This work was supported in part by the Agency of Industrial
Science and Technology grants and a Science and Technology
Agency/Center Of Excellence grant (to T. I.).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.
¶
Submitted as partial requirement for undergraduate study at
Tsukuba University.
§§
To whom all correspondence should be addressed: Biosignaling
Dept., National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Tel.: 81-298-54-6072; Fax:
81-298-54-6149; E-mail: imamura@nibh.go.jp.
The abbreviations used are:
FGF, fibroblast
growth factor;
SRP, signal recognition particle;
PCR, polymerase chain
reaction;
ER, endoplasmic reticulum;
DMEM, Dulbecco's modified
Eagle's medium;
PAGE, polyacrylamide gel electrophoresis;
PPL, preprolactin;
PBS, phosphate-buffered saline.
A Hydrophobic Region Locating at the Center of Fibroblast Growth
Factor-9 Is Crucial for Its Secretion*
§¶,
,,
, and§§
Biosignaling Department, School of Life Science,
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
PCR primers for full-length and deletion mutants of FGF-9
PCR primers for FGF-9 point mutants
SP-PL) were prepared as described previously (18). PPL and SP-PL
were transcribed using the mMESSAGE mMACHINE SP6, in vitro
transcription kit (Ambion Inc. Austin, TX). FGF-9 was linearized with
BamHI and transcribed using the mMESSAGE mMACHINE T3,
in vitro transcription kit (Ambion Inc.). Synthesized RNA
was translated in a wheat germ cell-free system containing
[3H]leucine (American Radiolabeled Chemicals, St. Louis)
in the presence or absence of purified SRP for 20 min at 26 °C. The
translated product was resolved by SDS-polyacrylamide gel
electrophoresis (PAGE). The gel was dried, immersed in fluorography
mixture (ENLIGHTNING, NEN Life Science Products), and exposed to Biomax
MS film (Eastman Kodak Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FGF-9 is efficiently secreted as a
glycosylated protein. COS cells transfected with an expression
vector encoding the FGF-9 cDNA were harvested (lane 1).
Equivalent aliquots of cell lysate (lane 1), conditioned
medium (lane 2), and N-glycanase-treated
conditioned medium (lane 3) were resolved by SDS-PAGE,
transferred to a membrane, and blotted with anti-FGF-9 monoclonal
antibody. The signal was visualized using peroxidase-labeled anti-mouse
IgG and ECL. Glycosylated and non-glycosylated forms of FGF-9 are
indicated by the closed and open arrowheads,
respectively. Positions of the molecular mass standards are indicated
by bars on the left.
SP-PL, which lacks a signal sequence, was unaffected by SRP. Interestingly, SRP clearly inhibited translation of FGF-9 in a manner
similar to PPL (Fig. 2). Thus, SRP appears to bind to a putative signal
sequence in FGF-9.
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Fig. 2.
In vitro translation of FGF-9 is
arrested by SRP. FGF-9, a mature form of prolactin
( SP-PL), and preprolactin (PPL) were
translated in vitro using a wheat germ translation system in
the presence of [3H]leucine and the indicated amounts of
SRP. The products were resolved by SDS-PAGE; the gel was then dried and
subjected to fluorography, and the fluorescent image was captured on
x-ray film. Positions of the molecular mass standards are indicated by
bars on the left.
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Fig. 3.
Translocation of FGF-9 and its deletion
mutants into microsomes. A, translocation of
full-length, wild-type FGF-9 into microsomes. FGF-9 was translated
in vitro in the presence of [3H]leucine using
reticulocyte lysate in the absence (lanes 1, 2, 5, and
6) or presence of microsomes (lanes 3, 4, 7, and
8) and in the absence (lanes 1, 3, 5 and
7) or presence of Triton X-100 (lanes 2, 4, 6,
and 8; TX-100). The translation products were
then digested with trypsin (lanes 1-4) or not treated
(lanes 5-8). The reaction products were resolved by
SDS-PAGE, and fluorography was performed as described in the legend to
Fig. 2. The glycosylated and non-glycosylated forms of FGF-9 are
indicated by closed and open arrowheads,
respectively. Positions of the molecular mass standards are indicated
by bars on the left. B-F, microsomal
translocation of various deletion mutants. B,
dFGF-9-(1-104); C, dFGF-9-(105-208); D,
dFGF-9-(53-156); E, dFGF-9-(1-156); and F,
dFGF-9-(91-208). Experiments were performed as in A, except
that the dFGF-9-(1-104), dFGF-9-(105-208), and dFGF-9-(53-156) were
precipitated by trichloroacetic acid prior to SDS-PAGE. Glycosylated
and non-glycosylated forms of the mutants are indicated as
closed and open arrowheads, respectively. The
arrow in F indicates a form of dFGF-9-(91-208)
after signal cleavage. G, diagram showing the various
deletion mutants aligned with the FGF-9 hydrophobic plot, as well as
the summarized results of their glycosylation and translocation.
Hydropathy plots were generated using the Kyte-Doolittle hydrophobic
scale and an interval of 10 amino acids. NGT indicates the
position of the N-glycosylation consensus sequence. The
asterisk denotes incapability of secretion. wt,
wild type.
Gly-Leu). Moreover, the
predicted molecular mass after secretion (11 kDa) was in agreement with
the present results. This means that when placed at the N terminus, the
central hydrophobic region of FGF-9 functions as a cleavable signal
sequence. These results, along with the observation that
FGF-9N33-(34-208) was neither secreted nor glycosylated (Fig. 5,
lanes 11 and 14), are summarized in Fig.
3G.
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Fig. 4.
Microsomal translocation of FGF-9 and its
point mutants. A, amino acid sequence of wild-type
(wt) rat FGF-9. Point mutation sites are depicted by the
letters beneath the main sequence. B, microsomal
translocation of full-length, wild-type FGF-9, and its point mutants.
Experiments were performed as described in the legend to Fig. 3. The
glycosylated and non-glycosylated forms of FGF-9 are indicated by
closed and open arrowheads, respectively.
Positions of the molecular mass standards are indicated by
bars on the left. C, hydropathy plots
of FGF-9 and its point mutants.
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[in a new window]
Fig. 5.
Analysis of processing and secretion of
various FGF-9 mutants expressed in COS transfectants. COS cells
were separately transfected with expression vectors encoding FGF-9
(lanes 1, 5, 9, and 12), FGF-9(L25N) (lanes
2 and 6), FGF-9(I100N, A101N) (lanes 3 and
7), FGF-9N33-(34-208) (lanes 11 and
14), dFGF-9-(91-208) (lane 15), or by vector
alone (lanes 4, 8, 10, and 13). After a period of
cell growth, the cells (lanes 1-4, 9-11, and
15) and the conditioned medium (lanes 5-8 and
12-14) were recovered and analyzed for the presence of
FGF-9 as described in the legend to Fig. 1. For comparison,
dFGF-9-(91-208) was also translated in vitro in the absence
of microsome (lane 16). The glycosylated and
non-glycosylated forms of each construct are indicated by
closed and open arrowheads, respectively. The
N-terminal cleaved form of dFGF-9-(91-208) is indicated by an
arrow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and 3 (signal peptides are numbered negatively from the
cleavage site toward the N terminus of the precursor; Ref. 25). The
weak hydrophobic region at its N terminus not withstanding, FGF-9 lacks
these characteristics, as confirmed by the signal sequence prediction
software. Indeed, the N terminus was intact in the secreted form of
FGF-9. Instead, based on SignalP analysis, the central hydrophobic
domain was predicted to serve as a cleavable signal sequence, a
prediction that was confirmed by our experimental results.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: National Research Institute of Vegetables,
Ornamental Plants and Tea.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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