(Received for publication, March 15, 1995; and in revised form, May 2, 1995)
From the
Insulin-like growth factor I (IGF-I) is a mitogenic peptide that
is produced in most tissues and cell lines and plays an important role
in embryonic development and postnatal growth. IGF-I is initially
synthesized as a prohormone precursor that is converted to mature IGF-I
by endoproteolytic removal of the carboxyl-terminal E-domain.
Regulation of the conversion of proIGF-I to mature IGF-I is a potential
mechanism by which the biological activity of this growth factor might
be modulated. Endoproteolysis of the IGF-I prohormone occurs at the
unique pentabasic motif
Lys-X-X-Lys-X-X-Arg
Insulin-like growth factor I (IGF-I)
Figure 2:
The proIGF-I-FLAG expression construct.
The 24 nucleotides coding for the 8-amino-acid FLAG peptide were
inserted between the codons for Ala
Figure 1:
The IGF-I prohormone processing site.
The amino acid sequence of the proIGF-I processing site is shown in single-letter code. Basic residues are indicated by boldface type and numbered. The scissile bond
connecting the P1 Arg
The paradigm established for
processing of proinsulin suggests that proIGF-I would be converted to
mature IGF-I by cleavage at the carboxyl terminus of Arg
Conditioned
medium (1 to 5 ml) was acidified with trifluoroacetic acid (0.1% final
concentration) and loaded onto a Sep-Pak C-18 cartridge (Millipore)
that had previously been equilibrated with 20% acetonitrile, 0.1%
trifluoroacetic acid. After washing the column in 20% acetonitrile,
0.1% trifluoroacetic acid, peptides were eluted in 80% acetonitrile,
0.1% trifluoroacetic acid, lyophilized, and reconstituted in 500 µl
of IP buffer. Immunoprecipitation was performed with 3 µg of M1 or
M2 anti-FLAG monoclonal antibody (Kodak/IBI) or 5 µl of UB3-189
anti-human somatomedin-C polyclonal antisera (National Hormone
Pituitary Program, NIDDK) overnight at 4 °C. M1 and M2 immune
complexes were precipitated with protein G-Sepharose (Pharmacia Biotech
Inc.), and UB3-189 immune complexes were precipitated with protein
A-Sepharose. The immunoprecipitates were washed twice in buffer
containing 25 mM Tris-Cl, pH 7.4, 300 mM NaCl, 1
mM CaCl
Immunoprecipitates were solubilized in SDS-sample
buffer containing 2-mercaptoethanol and denatured by heating at 95
°C for 5 min. Samples were electrophoresed on Tricine-buffered gels
with a 4% stack and a resolving gel of 10%, 16.5%, or 20%
polyacrylamide(20) . Gels were fixed in 10% acetic acid, 25%
isopropyl alcohol for 30 min, and then soaked in a fluorographic
solution (Amplify, Amersham) for 15 min. Dried gels were exposed to
x-ray film (Kodak) with an intensifying screen.
Before proceeding with biosynthetic
studies, it was necessary to determine if the presence of the FLAG
peptide would alter processing of the IGF-I prohormone. 293 cells were
transfected with IGF-I, R71X, and IGF-I-FLAG expression
vectors, and [
Figure 3:
The
FLAG peptide does not interfere with proIGF-I processing. 293 cells
were transfected with IGF-I, R71X, or IGF-I-FLAG expression
vectors, and [
Figure 4:
PreproIGF-I-FLAG is processed to mature
IGF-I-FLAG. 293 cells were transfected with R71X or IGF-I-FLAG
expression vectors, and [
Figure 5:
Multiple forms of unprocessed proIGF-I are
secreted. 293 cells were transfected with the IGF-I-FLAG expression
vector and metabolically labeled with
[
Figure 6:
ProIGF-I is N-glycosylated. 293
cells were transfected with the IGF-I-FLAG expression vector and
metabolically labeled with [
Figure 7:
Conversion of proIGF-I to mature IGF-I
occurs intracellularly. 293 cells were transfected with the R71X or IGF-I-FLAG expression vector, and
[
Fig. 8A shows the results of expression of the single substitution mutants
in 293 cells. On Tricine-buffered 20% polyacrylamide gels, the R71X mutant migrates as a single band. The R74X mutant
migrates as a poorly resolved doublet, and the R77X mutant
migrates as a clearly defined doublet. This gel electrophoresis system
is therefore capable of resolving IGF-I-FLAG peptides of 78, 81, and 84
amino acids in length. It is also interesting to note that both the
R74X and R77X peptides are processed at
Arg
Figure 8:
Processing of proIGF-I mutants in 293
cells. 293 cells were transfected with expression constructs coding for
wild-type (WT) and mutated proIGF-I-FLAG, as indicated: panel A, R71X, R74X, R77X, WT,
K68G, R71A, R74A, R77A; panel B, K65A/R71X, K65A,
K65A/R74A, K65A/R77A; panel C, K65A/K68G/R71X,
K65A/K68G; panel D, R77X, K68G/R71A, K68G/R74A,
K68G/R77A, R71A/R77A, R74A/R77A. Cells were metabolically labeled with
[
The K68G mutant processing pattern
is qualitatively similar to that of wild-type proIGF-I processing
except that all bands migrate slightly faster on Tricine-buffered gels (Fig. 8A). This band
shift may be attributed to size and/or charge differences of the lysine
and glycine side chains. When K68G and K68G/R71X peptides are
run together on Tricine-buffered 20% gels, it is clear that processing
does not occur at Arg
When Lys
Three K65A double mutants were
generated. The K65A/R74A and K65A/R77A mutants migrate as a single band
with the same molecular weight as the K65A/R71X mutant (Fig. 8B). These mutants are therefore cleaved at
Arg
While it was not
possible to definitively assign cleavage locations for the K68G double
mutants, it is clear that these mutants are processed very poorly, if
at all, at Arg
When CHO-K1 cells were transfected
with the R71X and R77X stop codon mutants, peptides
corresponding to cleavage products at Arg
Figure 9:
Processing of proIGF-I in RPE.40 cells.
CHO-K1 and RPE.40 cells were transfected with R71X,
R77X, or IGF-I-FLAG expression vectors and cells were
metabolically labeled with [
We have used the pCMVigf1-FLAG expression vector and human
embryonic kidney 293 cells to study proteolytic processing of human
proIGF-I. Using the UB3-189 anti-human IGF-I antisera, we have
shown that the pCMVigf1 and pCMVigf1-FLAG expression vectors direct
synthesis and secretion of processed IGF-I and IGF-I-FLAG, respectively (Fig. 3). Using the M1 and M2 monoclonal antibodies, we have
demonstrated that the FLAG epitope does not interfere with removal of
the signal peptide (Fig. 4). PreproIGF-I-FLAG is therefore
faithfully processed to mature IGF-I-FLAG and can be used to study
post-translation processing of proIGF-I.
When expressed in 293
cells, pCMVigf1-FLAG directs synthesis of multiple forms of high
molecular weight proIGF-I-FLAG peptides which are converted to
proIGF-I-FLAG by incubation with N-glycosidase ( Fig. 5and Fig. 6). Although glycosylated forms of human
proIGF-I have not been described previously, Simmons et al.(26) have shown that rat proIGF-I can be N-glycosylated in an in vitro translation system.
Interestingly, peptides generated from initiation of translation at
Met
Proprotein precursors can be converted to mature forms
intracellularly, at the cell surface membrane, or extracellularly.
Pulse-chase experiments have shown that the E-domain of proIGF-II is
cleaved at several sites during transport through the cell. Most of
these cleavages, including the final cleavage to produce the 7-kDa
mature IGF-II, appear to occur very late in the secretory pathway and
may also occur extracellularly(28) . We detect mature
IGF-I-FLAG in 293 cell lysates, indicating that conversion can occur
intracellularly (Fig. 7). We have not seen evidence for
processing of proIGF-I-FLAG to mature IGF-I-FLAG when proIGF-I-FLAG
peptides were incubated with conditioned media from 293 cells or serum
from mouse, rat, or fetal calves (data not shown).
Expression of
wild-type and mutant proIGF-I-FLAG constructs in 293 cells indicates
that the IGF-I prohormone can be cleaved at two sites, Arg
Since IGF-I isolated from
human serum is 70 amino acids long, it is likely that final maturation
occurs by cleavage of the precursor at Arg
The optimal SPC1 cleavage site has been defined as
RXK/RR (29) . SPC1 will also cleave some precursors
with an RXXR motif with reduced efficiency (15, 16, 29) while other precursors with this
motif are not cleaved(30) . It is unclear whether SPC1 will
cleave substrates containing a lysine residue in the P4 position. The
fusion glycoprotein precursor (F
It is interesting that the R74X and R77X stop codon mutants are cleaved at
Arg
We thank Dr. Graeme Bell for providing the pCMV6c
expression vector and Dr. Thomas Moehring for the CHO-K1 and RPE.40
cell lines. The UB3-189 polyclonal antisera was a gift from Dr. Terry
Taylor of the National Hormone and Pituitary Program. We also thank
Paul Gardner for synthesis of oligonucleotides. We are very grateful to
Drs. Shu Jin Chan, Yves Rouillé, and Kaare Lund for many helpful
discussions and comments on this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-X-X-Arg-X-X-Arg.
Recently, a family of enzymes which cleave prohormone precursors at
sites containing multiple basic residues has been discovered. The goals
of this study were 1) to determine which basic residues in the
pentabasic proIGF-I processing site were necessary for proper cleavage
and 2) to examine the role that subtilisin-related proprotein
convertase 1 (SPC1/furin) might play in proIGF-I processing. We have
shown that an expression vector coding for an epitope-tagged proIGF-I
directs synthesis and secretion of mature IGF-I-(1-70), extended
IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in
human embryonic kidney 293 cells. Extended IGF-I-(1-76) is
produced by cleavage at Arg
and requires both Arg
(P4) and Arg
(P1). Cleavage at Arg
does
not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting
that processing at this site is mediated by SPC1. Mature
IGF-I-(1-70) is produced by cleavage at Arg
and
requires both Lys
(P4) and Arg
(P1).
Lys
in the P7 position is important for efficient
cleavage. SPC1 is not required for processing at Arg
since
this cleavage occurs in RPE.40 and LoVo cells. These data suggest the
existence of a processing enzyme which is specific for the
Lys-X-X-Arg motif of proIGF-I.
(
)circulates in human serum as a 70-amino-acid peptide
consisting of four domains: B, C, A, and D. The physiological
importance of IGF-I for normal development and growth is underscored by
the severity of the phenotype of mice lacking a functional IGF-I gene.
Mice carrying a disrupted IGF-I gene display growth deficiencies,
delayed bone development, infertility, and a high mortality
rate(1, 2) . Liver is the main site of production of
circulating IGF-I, although this growth factor is also synthesized and
secreted by most tissues. Mature IGF-I can be derived from either of
two IGF-I prohormones by removal of the E-domain (see Fig. 2).
Alternative splicing of IGF-I mRNA is responsible for generating the
two IGF-I prohormones, and the physiological significance of the
different forms is unknown(3) . ProIGF-IA contains a
35-amino-acid E-domain, while the E-domain of proIGF-IB is 77 amino
acids in length. The sequences of human proIGF-IA and proIGF-IB are
identical through the first 16 residues of the E-domain(4) ,
including the unique pentabasic prohormone cleavage motif
Lys-X-X-Lys-X-X-Arg
-X-X-Arg-X-X-Arg (Fig. 1). This motif has been conserved in mammals, birds,
amphibians, and teleosts.
and
Gly
of the preproIGF-I cDNA, and the resulting
recombinant PCR product was cloned into the pCMV6c expression vector.
The CMV promoter and coding region for preproIGF-I-FLAG are boxed. SP, signal peptide; FLAG, FLAG
peptide; B, C, A, D, and E refer to the respective domains of proIGF-I. Numbering above the box indicates the position of amino acid residues
relative to the first amino acid of the B-domain. Cleavage sites for
signal peptidase and the proIGF-I convertase are indicated by vertical arrows. The R71X mutant was generated by
converting the codon for Arg
to a stop codon. The
monoclonal antibody M1 will recognize the FLAG peptide only when it is
located at the amino terminus of a protein while the M2 monoclonal
antibody will recognize the FLAG peptide in any location. Polyclonal
antisera UB3-189 was generated using mature human IGF-I as
antigen.
and the P1` Ser
is
indicated by the vertical arrow.
It is now well established that most
peptide hormones and growth factors are initially synthesized as
biologically inactive precursors that are converted to active forms by
endoproteolysis at specific sites. Proinsulin has served as a model for
studies of prohormone processing. From these studies it has been
determined that the C-peptide of proinsulin is excised by proteolytic
cleavage at paired dibasic residues to produce
insulin(5, 6) . Cleavage is mediated by two
subtilisin-related proprotein converting (SPC) enzymes, SPC2 (PC2) and
SPC3 (PC1/3)(7, 8) . These enzymes are serine
proteases and have been shown to process several prohormones to mature
hormones, including proglucagon (9) and the proopiomelanocortin
precursor(10, 11, 12) . Expression of SPC2
and SPC3 is limited to endocrine and neuroendocrine tissues, and the
preferred cleavage site is carboxyl-terminal to Arg-Arg and Lys-Arg
doublets(6) . Other members of the mammalian subtilisin-related
proprotein convertase family include SPC1 (furin), SPC4 (PACE4), SPC5
(PC4), and SPC6 (PC5/6). SPC1 and SPC4 are widely distributed and
cleave proprotein precursors at tri- and tetrabasic sites(13) .
The preferred cleavage site for SPC1 appears to be
Arg-X-Lys/Arg-Arg(14) , and the
Arg-X-X-Arg sequence may serve as a minimal
processing site(15) . SPC4 has been reported to have a more
strict requirement for a basic residue in the P2 position than does
SPC1(16, 17) .
,
followed by removal of the basic residue by a carboxypeptidase. SPC1 is
a candidate proIGF-I convertase since, like IGF-I, it is expressed
ubiquitously and it does not have a strict requirement for a basic
residue in the P2 position. We have used site-directed mutagenesis to
determine the importance of each of the basic residues in the proIGF-I
cleavage site for recognition by the processing enzyme. We have also
examined the role of SPC1 in proIGF-I processing through the use of
cell lines deficient in SPC1 activity.
Expression Constructs
PreproIGF-IA was amplified
by PCR from cDNA obtained from the human fibroblast cell line GM 03652C
(ATCC). Primers specific for the Met region of the
signal peptide (hIGF1-5) and the translation termination codon
region of the E-domain (hIGF1-6) were used:
5`-GGGAATTCTTGAAGGTGAAGATGCACAC and 5`-GGGGATCCCCTACATCCTGTAGTTCTTGT.
The PCR product was cloned into the EcoRI and BamHI
sites of the pCMV6c expression vector, which contains the CMV promotor
and the SV40 poly(A) tail, to generate pCMVigf1. pCMVigf1 was then used
as a template for recombinant PCR (18) to generate
pCMVigf1-FLAG. The signal peptide of IGF-I was amplified using primers
hIGF1-5 and Flg-2
(5`-CTTGTTCATCGTCGTCCTTGTAGTCAGCCGTGGCAGAGCTGGT). Flg-2 codes for
Thr
to Ala
of the signal peptide
and contains the 24-nucleotide antisense FLAG sequence on the 5` end.
The coding region of proIGF-I was amplified using primers hIGF1-6
and Flg-1 (5`-GACTACAAGGACGACGATGACAAGGGACCGGAGACGCTCTGC). Flg-1 codes
for Gly
to Cys
of the B-domain and contains the
24-nucleotide sense FLAG sequence on the 5` end. The PCR products were
purified by polyacrylamide gel electrophoresis, and 50 ng of each was
mixed, annealed, and extended under PCR conditions and amplified by 12
cycles of PCR with hIGF1-5 and hIGF1-6. The recombinant PCR
product was cloned into the EcoRI and BamHI sites of
pCMV6c to generate pCMVigf1-FLAG. The fidelity of PCR amplification was
verified by DNA sequencing.
Site-directed Mutagenesis
Site-directed mutants
were generated using the Altered Sites II in vitro Mutagenesis
System (Promega) and double-stranded proIGF-I-FLAG plasmid DNA as
template. Oligonucleotide primer sequences and corresponding templates
are shown in Table 1. Mutants were subcloned from pAlter to
pCMV6c and verified by DNA sequencing.
Cell Culture and Transfections
Human embryonic
kidney 293 cells were grown in DMEM (Life Technologies, Inc) with 10%
fetal calf serum and 100 units/ml penicillin and 100 µg/ml
streptomycin. 293 cells were transfected by the calcium phosphate
method(19) . CHO-K1 and RPE.40 cells (provided by Dr. Thomas
Moehring, University of Vermont) were grown in Ham's F-12 medium
with 5% fetal calf serum and 100 units/ml penicillin and 100 µg/ml
streptomycin. CHO-K1 and RPE.40 cells were transfected using
LipofectAMINE following the manufacturer's protocol
(Life Technologies, Inc).
Cell Labeling, Immunoprecipitation, and Gel
Electrophoresis
24 h after transfection cells were washed twice
in phosphate-buffered saline and incubated for 2 h in cysteine-free (or
histidine-free) Dulbecco's modified Eagle's medium with 1
mg/ml bovine serum albumin, 100 units/ml penicillin, and 100 µg/ml
streptomycin. The medium was then replaced with cysteine-free (or
histidine-free) Dulbecco's modified Eagle's medium
containing 100 µCi/ml [S]cysteine (Amersham,
1000 Ci/mmol) or [
C]histidine (DuPont NEN, 385
Ci/mmol), 1 mg/ml bovine serum albumin, 100 units/ml penicillin, and
100 µg/ml streptomycin. After labeling for 24 h, conditioned medium
was collected and centrifuged to remove cellular debris. Conditioned
medium was stored at -20 °C. Cells were washed twice with
phosphate-buffered saline and removed from the plate in
immunoprecipitation (IP) buffer (25 mM Tris-Cl, pH 7.4, 300
mM NaCl, 1 mM CaCl
, 10 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% Triton
X-100), sonicated, and stored at -20 °C.
, and then once in buffer consisting of 25
mM Tris-Cl, pH 7.4, 140 mM NaCl, 1 mM CaCl
. Cell lysates were thawed and centrifuged to
remove cell debris. 500 µl of supernatant was preabsorbed with
protein G-Sepharose and immunoprecipitated with M1 antibody as
described above.
Deglycosylation
7 ml of conditioned medium from
[S]cysteine-labeled cells was concentrated on a
Sep-Pak and lyophilized as described above. The lyophilized proteins
were solubilized in 0.1% SDS, denatured by heating at 95 °C for 10
min, and placed on ice. 77 µl of 10
deglycosylation buffer
(200 mM NaH
PO
, pH 7.2, 5% Nonidet
P-40) was added, and 100-µl aliquots were incubated with 2
milliunits of neuraminidase (sialidase), 2.5 milliunits of O-glycosidase, 0.4 unit of N-glycosidase F
(Boehringer Mannheim), or combinations of these enzymes for 24 h at 37
°C. After incubation, 400 µl of IP buffer was added, and
epitope-tagged peptides were immunoprecipitated with M1 antibody and
run on 10% polyacrylamide gels as described above.
The FLAG Peptide Does Not Interfere with Processing of
PreproIGF-I
Our initial attempts to study the processing of
proIGF-I were hampered by difficulties in immunoprecipitating proIGF-I
from conditioned medium. Commercially available antisera to IGF-I have
been generated using mature IGF-I as antigen, and the E-domain of
proIGF-I may interfere with epitope recognition. To circumvent this
problem we designed a preproIGF-I expression vector that contained an
epitope tag, the 8-amino-acid FLAG peptide, between Ala of the signal peptide and Gly
of the B-domain (Fig. 2). Two monoclonal antibodies to the FLAG peptide have
been generated. The M1 antibody requires calcium for binding and will
only recognize a FLAG peptide with an exposed amino terminus. The M1
antibody can therefore be used to determine whether the signal peptide
has been removed from proIGF-I-FLAG. The M2 antibody will recognize the
FLAG peptide in any location and should therefore immunoprecipitate all
epitope-tagged peptides. To obtain an accurate molecular weight marker
for IGF-I-FLAG, we mutated Arg
of proIGF-I-FLAG to a stop
codon. This R71X mutant directs synthesis of the 70-amino-acid
mature IGF-I molecule with the FLAG peptide at the amino terminus ( Fig. 2and Table 1).
S]cysteine-labeled proteins were
immunoprecipitated from conditioned medium using UB3-189 anti-human
IGF-I antisera (Fig. 3). Two proteins of 7-8 kDa were
immunoprecipitated from conditioned media of cells transfected with
CMVigf1. Two proteins were also immunoprecipitated from conditioned
media of cells transfected with CMVigf1-FLAG. These peptides have a
molecular mass of 9-10 kDa and represent the epitope-tagged
homologues of the IGF-I peptides synthesized from the CMVigf1 vector.
The 9-kDa band generated from the IGF-I-FLAG transfection migrates at
the same position as the R71X mutant, which has a predicted
molecular mass of 8640 daltons, indicating that proIGF-I-FLAG is
processed at Arg
. The larger molecular mass bands of the
IGF-I and IGF-I-FLAG doublets may represent carboxyl-extended forms of
mature IGF-I.
S]cysteine-labeled IGF peptides
were immunoprecipitated using the UB3-189 antisera and resolved on
Tricine-buffered 16.5% polyacrylamide gels, as described under
``Experimental Procedures.'' M indicates mock
transfection. The molecular masses of the
C-methylated
protein markers are indicated.
When conditioned medium from R71X-transfected
293 cells was immunoprecipitated with M1, M2, and UB3-189 antisera, a
predominant band was seen at 9 kDa (Fig. 4). The slightly larger
band recognized by the M1 antibody may be an oxidized form of IGF-I.
When conditioned medium from IGF-I-FLAG-transfected 293 cells was
immunoprecipitated with these antisera, several peptides were
visualized. The 9-kDa band generated from the R71X transfections was also present in IGF-I-FLAG-transfected media and
was recognized by anti-FLAG and anti-human IGF-I antisera, indicating
that this band represents mature IGF-I-FLAG. The 10-kDa band from the
IGF-I-FLAG-transfected media was also recognized by all three antisera
and may represent a carboxyl-extended form of mature IGF-I-FLAG. In
addition to the 9-10-kDa doublet, the M1 and M2 antibodies
immunoprecipitated peptides of 14 kDa and 16 kDa, as well as a broad
band from 19-21 kDa. Since these peptides were recognized by both
M1 and M2, they do not represent forms of IGF-I containing a signal
peptide and thus the FLAG peptide does not interfere with signal
peptidase activity. The higher molecular weight bands, which were not
immunoprecipitated with the UB3-189 antisera, may be forms of proIGF-I.
S]cysteine-labeled
peptides were immunoprecipitated from aliquots of reconstituted
conditioned medium with M1, M2, or UB3-189 antisera. Immunoprecipitates
were resolved on Tricine-buffered 16.5% polyacrylamide gels, as
described under ``Experimental Procedures.'' The molecular
masses of the
C-methylated protein markers are
indicated.
ProIGF-I and N-Glycosylated ProIGF-I Are Secreted by
Transfected 293 Cells
Selective amino acid labeling was employed
in order to determine whether the high molecular weight proteins
immunoprecipitated from IGF-I-FLAG-transfected conditioned media by
anti-FLAG antibodies were unprocessed or partially processed proIGF-I
molecules. Human proIGF-IA contains two histidine residues, both of
which are located in the E-domain. Therefore, histidine labeling should
be specific for proIGF-I as opposed to oligomers or aberrantly
processed mature IGF-I. Fig. 5shows the results of
[S]cysteine and
[
C]histidine labeling of cells transfected with
the IGF-I-FLAG expression vector. The 9-10-kDa doublet migrated
as a single band on this 10% polyacrylamide gel and was clearly visible
when cells were labeled with [
S]cysteine but was
not present when labeled with [
C]histidine. In
contrast, 14-kDa and 19-21-kDa proteins were visible from both
[
S]cysteine and
[
C]histidine labeling, and the
[
C]histidine-labeled 16-kDa band could be seen
after overexposure (data not shown).
S]cysteine (Cys) or
[
C]histidine (His). Peptides were
immunoprecipitated from reconstituted conditioned medium with the M2
antibody and resolved on Tricine-buffered 10% polyacrylamide gels. M = mock, T = transfected. The
molecular masses of
C-methylated protein markers are
indicated.
The predicted molecular mass of
proIGF-I-FLAG is 12.7 kDa, in close agreement with the 14-kDa band seen
in [C]histidine labeling. The proIGF-IA E-domain
contains one potential N-glycosylation site at Asn
and several serine and threonine residues which could be used for O-glycosylation. To determine if the 16-kDa and
19-21-kDa peptides were glycosylated forms of proIGF-I, peptides
from conditioned medium of proIGF-I-FLAG-transfected 293 cells were
subjected to treatment with various glycosidases. As shown in Fig. 6, neuraminidase and O-glycosidase had no effect
on the electrophoretic mobility of proIGF-I peptides. However, the
19-21-kDa band completely disappeared after digestion with N-glycosidase while the intensity of the 14-kDa and 16-kDa
peptides increased dramatically. It is therefore likely that the
19-21-kDa peptide is N-glycosylated proIGF-I-FLAG, the
14-kDa peptide is proIGF-I-FLAG, and the 16-kDa peptide may be
partially glycosylated proIGF-I-FLAG.
S]cysteine.
Conditioned medium was concentrated on a Sep-Pak, lyophilized,
reconstituted, and denatured. One-tenth volume of 10
deglycosylation buffer was then added, and aliquots were incubated in
the absence of enzyme(-), or with neuraminidase (S), O-glycosidase (O), N-glycosidase (N) alone or in combinations for 24 h at 37 °C. Peptides
were immunoprecipitated with M1 antibody and run on Tricine-buffered
10% polyacrylamide gels. The molecular masses of the
C-methylated protein markers are
indicated.
Conversion of ProIGF-I to Mature IGF-I Occurs
Intracellularly
Since there are examples of growth factors that
can be proteolytically activated intra- or extracellularly, it was of
interest to determine the location of conversion of proIGF-I to mature
IGF-I. Cell lysates from R71X or IGF-I-FLAG-transfected 293
cells were immunoprecipitated with the M1 antibody, and peptides were
resolved on 16.5% polyacrylamide gels. The 9-10-kDa processed
IGF-I-FLAG doublet, as well as proIGF-I-FLAG and glycosylated
proIGF-I-FLAG, were clearly visible in cell lysates, indicating that
conversion can occur intracellularly (Fig. 7). We have also
demonstrated that proIGF-I-FLAG peptides are not processed to mature
IGF-I-FLAG when incubated with conditioned media from 293 cells or 50%
fetal calf, rat, or mouse serum for up to 24 h at 37 °C. However,
degradation of proIGF-I-FLAG occurs when incubated with trypsin (data
not shown).
S]cysteine-labeled peptides were
immunoprecipitated from cell lysates with the M1 antibody and resolved
on Tricine-buffered 16.5% polyacrylamide gels, as described under
``Experimental Procedures.'' M indicates mock
transfection. The molecular masses of the
C-methylated
protein markers are indicated.
Processing of ProIGF-I Mutants in 293 Cells
To
determine the importance of each of the basic residues in the
pentabasic proIGF-I processing motif for prohormone conversion
activity, we systematically mutated each basic residue to an uncharged
residue (Table 1). Single, double, and triple mutants were
generated. Since our initial experiments with the R71X and
IGF-I-FLAG expression vectors indicated that proIGF-I was being cleaved
at Arg and a downstream site, we also made two additional
stop codon mutants for use as molecular weight markers. Arg
and Arg
were mutated to stop codons, generating
R74X and R77X, respectively. In several cases,
substitution of a basic residue with an uncharged amino acid changed
the electrophoretic mobility of the protein. In some of these instances
it was necessary to make additional stop codon mutants in order to
correctly identify the processing site.
, indicating that as few as two residues on the
carboxyl terminus of Arg
are sufficient for processing
activity.
S]cysteine, and peptides were
immunoprecipitated with the M1 antibody. Immunoprecipitates were
resolved on Tricine-buffered 20% polyacrylamide gels, as described
under ``Experimental Procedures.'' The molecular masses of
the
C-methylated protein markers are
indicated.
As shown previously ( Fig. 3and Fig. 4),
wild-type IGF-I-FLAG is processed at two sites yielding a peptide that
migrates with the R71X standard at 9 kDa as well as a 10-kDa
peptide. Comparison of these peptides with the pattern produced by
expression of R74X and R77X indicates that the 10-kDa
IGF-I-FLAG peptide is generated by cleavage at Arg (Fig. 8A). In several different transfection
experiments, we have observed that approximately 50% of proIGF-I-FLAG
is cleaved at Arg
and 50% at Arg
. We have not
observed evidence for cleavage at Arg
(Table 2).
Mutation of Arg to alanine (R71A) abolishes processing
at this site, indicating that the basic residue is required at the P1
site. Processing at Arg
is not qualitatively affected (Fig. 8A). Mutations at either Arg
(R74A)
or Arg
(R77A) eliminate processing at Arg
without qualitatively affecting cleavage at Arg
(Fig. 8A).
. The K68G peptide migrates more
slowly than the K68G/R71X stop codon mutant (data not shown)
and is thus processed at a downstream site such as Arg
.
The P4 lysine residue is therefore required for processing at
Arg
.
is mutated to Ala (K65A),
the peptide is processed at two sites (Fig. 8B). The
smaller band migrates with the K65A/R71X mutant, indicating
that it is generated by cleavage at Arg
. Since the larger
band of the doublet is well resolved, it is likely that it represents
processing at Arg
.
but not Arg
. The processed K65A/K68G
mutant has a higher molecular weight than the corresponding
K65A/K68G/R71X mutant, indicating that the two upstream lysine
residues are required for cleavage at Arg
(Fig. 8C). It is likely that cleavage of
K65A/K68G occurs at Arg
since the K65A/K68G and
K65A/K68G/R71X bands are clearly resolved.
. Only 10% of the K68G/R74A mutants and 16%
of the K68G/R77A mutants were processed (Fig. 8D and Table 2). 79% of the K68G/R71A mutant was processed, and it is
likely that cleavage occurred at Arg
since this site is
preferred to Arg
in proIGF-I-FLAG. When Arg
is isolated by substitution of alanine for Arg
and
Arg
(R71A/R77A), only 8% of the precursor is cleaved. When
Arg
and Arg
are changed to alanine residues
(R74A/R77A), approximately 50% of the precursor is cleaved at
Arg
(Fig. 8D and Table 2).
Processing of ProIGF-I in RPE.40 Cells
The data on
processing of proIGF-I mutants indicated that processing at Arg was prevented in all mutants having a substitution at either
Arg
or Arg
( Fig. 8and Table 2),
suggesting that this sequence might be a cleavage site for SPC1, which
has minimally been defined as RXXR(15) . These data
also demonstrate that processing at Arg
requires both
Lys
(P4) and Arg
(P1). It is not yet clear if
SPC1 will process precursors containing a Lys for Arg substitution in
the P4 position. In order to determine if SPC1 is involved in proIGF-I
processing, we expressed IGF-I-FLAG in CHO-K1 and RPE.40 cells. RPE.40
cells were derived from CHO.K1 cells exposed to Pseudomonas exotoxin A (PEA) and selected for resistance (21) . RPE.40
cells fail to process precursor membrane glycoproteins of several
viruses, as well as the insulin proreceptor. This processing deficiency
can be corrected by transfection with SPC1
cDNA(22, 23) .
and Arg
were immunoprecipitated from conditioned media (Fig. 9).
Transfection with IGF-I-FLAG produced the Arg
and
Arg
doublet, as well as proIGF-I and glycosylated
proIGF-I. Expression of R71X and R77X in RPE.40 cells
resulted in the same pattern of immunoprecipitable peptides as was seen
in transfected CHO-K1 cells. However, when IGF-I-FLAG was expressed in
RPE.40 cells, a single band of mature IGF-I, corresponding to
IGF-I-FLAG processed at Arg
, was observed. This indicates
that RPE.40 cells process proIGF-I at Arg
but not
Arg
. We have also observed a single band corresponding to
Arg
cleavage when IGF-I-FLAG was transfected into LoVo
cells (data not shown). LoVo cells do not process the MET protooncogene
or the insulin proreceptor(24) , and this processing deficiency
has been attributed to a defective SPC1 gene (25) .
S]cysteine. Peptides
were immunoprecipitated with the M1 antibody and resolved on
Tricine-buffered 16.5% polyacrylamide gels, as described under
``Experimental Procedures.'' M indicates mock
transfection. The molecular masses of the
C-methylated
protein markers are indicated.
or Met
, but not
Met
, were substrates for N-glycosylation.
The pCMVigf1-FLAG expression vector contains translation initiation
codons at Met
and Met
, but
Met
has been eliminated. N-Glycosylated
proIGF-I has not been identified in vivo, and the significance
of this post-translational modification is unknown. However, it has
been demonstrated that glycosylation can affect proteolytic processing
of some precursor proteins. For instance, N-glycosylation of
the human influenza virus hemagglutinin precursor at Asn
can inhibit processing at Arg
(27) .
and Arg
. The Arg
cleavage site conforms
to the minimal SPC1 cleavage site defined by Molloy et al.(15) with Arg
as the P4 residue and
Arg
as the P1 residue. All mutants containing alanine in
place of Arg
or Arg
eliminate processing at
Arg
(R74A, R77A, K65A/R74A, K65A/R77A, K68G/R77A,
R71A/R77A, R74A/R77A). In all mutants containing substitutions of
nonbasic residues for Lys
, Lys
, or
Arg
, the percentage of precursor processing at Arg
is increased (K65A, K68G, R71A, K65A/K68G, K68G/R71A) (Table 2). One explanation for the increased processing at
Arg
in these mutants is that the secondary structure of
the peptide may have been modified in a way that allows for more
favorable enzyme-substrate interactions. However, each of the
Lys
, Lys
, and Arg
mutants also
decreases or eliminates processing at Arg
. It is therefore
likely that the increased processing at Arg
is a result of
the creation of a less efficient Arg
cleavage site. In
addition to mutational analysis, data obtained from the expression of
wild-type proIGF-I-FLAG in SPC1-deficient cell lines also implicate
SPC1 as the Arg
cleavage enzyme. When wild-type
proIGF-I-FLAG is expressed in CHO-K1 cells, cleavage occurs at
Arg
and Arg
. However, when expressed in
RPE.40 cells, a CHO-K1 derivative lacking SPC1 activity, cleavage of
proIGF-I-FLAG occurs at Arg
but not Arg
(Fig. 9). Processing at Arg
, but not
Arg
, was also observed when proIGF-I-FLAG was expressed in
the SPC1-deficient LoVo cell line (data not shown). It therefore
appears that SPC1 is required for cleavage of proIGF-I-FLAG at
Arg
but not Arg
.
. The presence
of a second cleavage site at Arg
, which affects processing
efficiency at Arg
, makes it difficult to determine the
minimal processing motif necessary for final maturation. Mutational
analysis has revealed that Lys
and Arg
are
both essential for processing at Arg
. Substitution of
glycine for Lys
(K68G) or alanine for Arg
(R71A) eliminates processing at Arg
(Fig. 8A, Table 2). The P7 lysine residue
may also be important for efficient processing since 28% of the K65A
mutant is cleaved at Arg
, as opposed to 55% for wild-type
proIGF-I. It thus seems paradoxical that the K65A/R74A and K65A/R77A
double mutants are processed more efficiently at Arg
than
the K65A single mutant (68% and 92%, respectively) (Table 2). A
likely explanation, however, is that the double mutants eliminate the
P4 and P1 residues, respectively, of the SPC1 cleavage site, therefore
making more substrate available for the Arg
cleaving
enzyme. Arg
alone is not sufficient for processing since
the K68G/R74A mutant is cleaved very poorly (Fig. 8D, Table 2).
) of the Ulster strain of
Newcastle disease virus contains the KQGR cleavage motif. The F
glycoprotein of Newcastle disease virus strain Ulster was not
cleaved when coexpressed or incubated in vitro with
SPC1(30) . A virulent avian influenza virus hemagglutinin
containing the RKKR cleavage motif was processed efficiently by SPC1 in
both coexpression and in vitro incubation experiments. When
the P4 arginine of this motif was mutated to lysine to create KKKR, 75%
of the hemagglutinin was cleaved when coexpressed with SPC1, and the
hemagglutinin was not cleaved when incubated with SPC1 in
vitro(31) . Our data on expression of proIGF-I-FLAG in
RPE.40 and LoVo cells indicate that SPC1 is not necessary for final
maturation of IGF-I. The identity of the proIGF-I converting enzyme is
unknown. SPC4 is a candidate because it is expressed in many tissue
types. However, this enzyme apparently has a more strict requirement
for a basic residue in the P2 position than does
SPC1(16, 17) . For many proprotein precursors that are
processed at basic residues, the scissile bond is on the carboxyl
terminus of the P1 residue. After cleavage of the scissile bond by a
specific endoprotease, the basic residues are removed by
carboxypeptidases. Recently, a metalloendopeptidase which cleaves
peptide bonds on the amino terminus of arginine residues was cloned
from a rat testis cDNA library(32) . Although the proIGF-I
cleavage site is similar to the motif recognized by enzymes of the SPC
family, it is possible that a different class of enzyme may convert
proIGF-I to mature IGF-I.
. This indicates that as few as two amino acids on the
carboxyl terminus of the P1 residue are sufficient for precursor
processing by the enzyme. Processing of these mutants, as well as
mutants containing alanine substitutions at Arg
or
Arg
, also demonstrates that cleavage at Arg
need not precede cleavage at Arg
. Pulse-chase
experiments have shown that proIGF-II is cleaved at multiple sites in
the E-domain(28) . Similar experiments will be necessary to
determine if proIGF-I is cleaved at Arg
before final
maturation. At this time there is no evidence for the existence of
IGF-I-(1-77) in vivo and the SPC1-mediated cleavage
observed at Arg
in our experiments may be a consequence of
overexpression. Nonetheless, this observation is interesting in terms
of SPC site selection specificity, and proIGF-I may be a useful model
for studying this aspect of prohormone processing. Three repetitive
motifs of basic-X-X-basic residues are contained
within only 10 amino acids. SPC1 recognizes the motif
R
XXR
. The motif
K
XXR
may also be recognized by SPC1,
but it is likely to be cleaved by another converting enzyme as well.
The R
XXR
motif does not appear to be
recognized by SPC1 or the proIGF-I converting enzyme. In fact, this
site appears to be highly refractory to cleavage. Attempts to force
cleavage at Arg
by isolating the
R
XXR
(K68G/R77A) or the single
Arg
(R71A/R77A) were unsuccessful (Fig. 8D, Table 2). Knowledge of the role of
nonbasic residues in proIGF-I processing, or the secondary structure of
the pentabasic motif, could provide useful insights into processing
specificity of prohormone convertases.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.