Expression and characterization of VIP and two VIP mutants in NIH 3T3 cells.
ABSTRACT Prepro-vasoactive intestinal peptide (prepro VIP) was expressed in NIH 3T3 cells, and the prepro VIP-derived peptides produced by the cells were analyzed by chromatography combined with sequence-specific radio-immunoanalysis. In accordance with what has previously been reported on processing in non-endocrine cell lines, the VIP precursor was processed poorly in these non-endocrine cells. Mainly an extended form of VIP could be detected in the media from the cells, and no immunoreactivity specific for amidated VIP was found. However, by changing the dibasic cleavage site positioned N-terminal to the VIP sequence in the precursor into the consensus sequence (Arg, X,Lys/Arg,Arg) for the ubiquitous processing enzyme furin, thought to process, e.g. insulin receptors, factor VII, and by deleting residues 156-170 in the VIP precursor, expression of amidated VIP was obtained in this fibroblast cell line. Peptides from the wild-type VIP precursor as well as peptides from the mutated VIP precursor were found to be able to stimulate the adenylate cyclase in cells expressing the VIP receptor.
- [Show abstract] [Hide abstract]
ABSTRACT: In many ganglia, the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) innervates nerve cell bodies containing the homologous neuropeptide vasoactive intestinal polypeptide (VIP). We therefore investigated whether PACAP affected the VIP gene expression and elucidated the molecular mechanisms using the human neuroblastoma cell line NB-1. A concentration dependent induction of the VIP mRNA level was found upon PACAP stimulation. Five nM PACAP mediated transient elevation of the VIP mRNA being evident after 2 h, the maximal 65-fold induction was reached after 6-8 h and hereafter the level decreased rapidly. In cell extracts, the concentration of immunoreactive VIP was elevated four-fold upon PACAP stimulation for 8 h, and it remained elevated during the next 40 h. In conditioned medium, a stable 20-fold VIP increase was seen after 8-24 h. Experiments with the translational inhibitor cycloheximide showed a direct effect of PACAP on the VIP mRNA level, and nuclear run-on assays revealed a three- to four-fold enhancement of the VIP gene transcription rate after PACAP stimulation. The VIP mRNA induction was abolished by transcriptional inhibition with the actinomycin D, and PACAP did not seem to mediate any changes in the VIP mRNA half-life. However, the VIP mRNA level seemed very stable during the transcriptional cessation. Reporter gene constructs were used to evaluate involvement of the VIP CRE site in the PACAP mediated induction of the VIP gene transcription. Mutation of the CRE site did not abolish the induction suggesting it to be of minor if any importance for the induction. In conclusion, the PACAP mediated induction of the VIP gene expression suggests that PACAP released from nerve terminals could influence the function of VIP'ergic neurons in target tissues.Molecular Brain Research 07/2000; 79(1-2):67-76. · 2.00 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The pharmacological profile of adenylyl cyclase activity was analysed in WI-38 human foetal lung fibroblasts. Among various agents that act through G-protein coupled receptors, only the beta-adrenergic agonist isoproterenol stimulated and the tetradecapeptide somatostatin (SRIF, sst) inhibited the enzyme activity. The use of the reverse transcription-polymerase chain reaction (RT-PCR) methodology with appropriate cDNAs allowed us to identify the expression of four subtypes of SRIF transmembrane receptors (sst1-4 but not sst5 receptors) in this cell line. By RT-PCR and immunochemistry techniques, we also demonstrated the expression of stimulatory (alpha(s)) and inhibitory (alpha(i1), alpha(i2) and alpha(i3)) G-protein subunits. The known role of the adenylyl cyclase system in cell proliferation and differentiation mechanisms together with the present analysis of the corresponding regulatory network in fibroblasts of human foetal lung add knowledge on the cell line WI-38 that is widely used as a model system in studying cell growth. The importance of this cell class in normal and abnormal lung function and development reinforces the significance of these results.Regulatory Peptides 12/2000; 95(1-3):53-8. · 2.06 Impact Factor
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ABSTRACT: To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are expressed in the suprachiasmatic nucleus (SCN), and in agreement with VIP signaling being crucial for maintenance of rhythmicity, we found both VIP and VPAC2 mRNA increased after differentiation of PC12 cells. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts time- and concentration-dependent effects on Per gene expression in the SCN. We added 1 nM and 1 microM PACAP to oscillating PC12 cells at times corresponding to midday and early and late night to evaluate whether the effects were similar as in SCN. Induction of Per1 mRNA was found at all three times, which differs from results in SCN. Thus, PC12 cells seem more useful for studying mechanisms behind acquirement of rhythmicity of cell cultures than for resetting of circadian rhythm.Journal of Molecular Neuroscience 10/2008; 36(1-3):132-40. · 2.89 Impact Factor
LET T ERS
FEBS Letters 341 (1994) 4348
Expression and characterization of VIP
NIH 3T3 cells
and two VIP mutants in
Birgitte S. WulfP, Birgitte Georg, Jan Fahrenkrug
University Department of Clinical Biochemistry, Bispebjerg Hospital, Bispebjerg bakke 23. DK-2400 Copenhagen NY Denmark
Received 24 January 1994
Prepro-vasoactive intestinal peptide @reproVIP) was expressed in NIH 3T3 cells, and the preproVIP-derived peptides produced by the cells were
analyzed by chromatography combined with sequence-specific radio-immunoanalysis.
processing in non-endocrine cell lines, the VIP precursor was processed poorly in these non-endocrine cells. Mainly an extended form of VIP could
be detected in the media from the cells, and no immunoreactivity specific for amidated VIP was found. However, by changing the dibasic cleavage
site positioned N-terminal to the VIP sequence in the precursor into the consensus sequence (Arg,X,Lys/Arg,Arg) for the ubiquitous processing
enzyme furin, thought to process, e.g. insulin receptors, factor VII, and by deleting residues 156170 in the VIP precursor, expression of amidated
VIP was obtained in this fibroblast cell line. Peptides from the wild-type VIP precursor as well as peptides from the mutated VIP precursor were
found to be able to stimulate the adenylate cyclase in cells expressing the VIP receptor.
In accordance with what has previously been reported on
Key words: Vasoactive intestinal peptide; Prohormone processing; NIH 3T3; Amidation
Vasoactive intestinal polypeptide (VIP) is a 28 amino
sacid neuropeptide with a broad range of biological ac-
tivities. The precursor for VIP (preproVIP) is synthe-
sized as a 170 amino acid polypeptide, which after re-
moval of the signal peptide is proteolytically
at mono and dibasic cleavage sites into a N-terminal
flanking peptide (preproVIP 22-79), PHM (peptide with
N-terminal His and C-terminal Met-amide), preproVIP
111-122, VIP, and a C-terminal flanking peptide (pre-
proVIP 1% 170), as shown in Fig. 1. The dibasic amino
acids C- terminal to the VIP and PHM sequences are
removed with a carboxypeptidase
C-terminal glycine residue in PHM and VIP is converted
by peptidyl-glycine a-amidating monooxygenase (PAM)
to amidated PHM and VIP. In some instances the dibasic
cleavage site C-terminal to PHM is left uncleaved result-
ing in a C-terminal extension of PHM to a 42 amino acid
peptide, PHV (peptide with N-terminal His and C-termi-
nal Val) (Fig. 1). The ratio of PHM to PHV is 1:l in
gastric antrnm and 9: 1 in colon and brain, and thus this
alternative processing is tissue specific. [ 1,2]. In the pres-
ent paper, we report the expression of cDNA encoding
preproVIP in a non-endocrine cell line, NIH 3T3. These
cells lack secretory granules, and, as expected, the pre-
H-like activity and the
*Corresponding author. Present address: Department of Molecular
Pharmacology, 1 CS36, Novo-Nordisk, DK-2880 Bagsvaerd, Denmark.
Fax: (45) 44 98 50 07.
cursor was poorly processed. In an attempt to obtain
efficient processing of the cleavage site positioned N-
terminal to the VIP sequence in the precursor, the resi-
dues at position 121 and 122 were both changed into
Arg, resulting in a tetrabasic cleavage site. This corre-
sponds to the consensus
Arg,Arg) for the ubiquitous processing enzyme furin,
which processes a number proproteins e.g. the insulin
receptor, factor VII and factor IX [3-51. Previously it was
reported that when a C-terminal truncated NPY (NPY-
Gly,Lys,Arg) is expressed in non-endocrine
cells synthesize amidated NPY . In order to probe the
possibility of expressing amidated VIP in NIH 3T3 cells,
a truncated VIP precursor with residues 15&l 70 deleted
was constructed. This truncation was made on the tetra-
basic VIP mutant precursor resulting in a VIP precursor
with a tetrabasic cleavage site N-terminal to the VIP
sequence and with only Gly,Lys,Arg as the C-terminal
extension of VIP. With this mutant it was possible to
obtain expression of amidated VIP in the NIH 3T3 cell
line, indicating that non-endocrine cell lines might be a
promising alternative to chemical synthesis for produc-
tion of amidated or otherwise
hormones and neuropeptides.
cleavage site (Arg,X,Lys/
2.1. DNA construction
The VIP cDNA, (kindly provided by Professor H. Okamoto, Tohuko
University School of Medicine, Sendai 980, Japan), lacked the se-
0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved.
B.S. Wurff et al. IFEBS Letters 341 (1994) 4348
quences encoding the precursor signal sequence. In order to obtain a
full-length VIP cDNA the missing part was obtained by PCR on human
genomic DNA. After verification of the PCR fragment by sequence
analysis, the fragment was subcloned into the VIP cDNA resulting in
a full length cDNA. The 5’ PCR primer introduced at the same time
a BamHI restriction enzyme site 5’ to the initiation codon.
To obtain expression of the VIP cDNA in mammalian cells, an
expression vector containing the human growth hormone polyadenyla-
tion signal and a human ubiquitin promoter was constructed in the
following way: the promoter from the human ubiquitin C gene  was
subcloned as a Hind111 fragment into HindIII-digested pUC19 resulting
in pHD183. A Klenow-treated SmaI-EcoRI fragment from pHD184
 containing the human growth hormone polyadenylation signal was
subcloned into a blunt-ended EcoRI-digested pHD183. This resulted
in the expression vector pBW87. A 1,130 bp BamHI-DraI VIP cDNA
fragment was rendered blunt by Klenow polymerase and subcloned
into blunt-ended BamHI-digested pBW87 resulting in the preproVIP
expression vector pBW91. By using the DruI restriction enzyme site
in the VIP cDNA, the CC tailing which arose from the original
cloning of the VIP cDNA  was excluded. The two mutants:
[PV121,122RR]preproVIP (tetrabasic mutant) and [PV121,122RR,
d156170lpreproVIP (tetrabasic, 15&170-deletion mutant) were intro-
duced by site-directed mutagenesis by the Altered sites in vitro mut-
agenesis system (Promega, Wisconsin, USA) and the mutagenic primers
5’-AGT GAC GTT TGC GAC GTA CAG GGT CT-3’ for the intro-
duction of arginine in residues 121 and 122, and 5’-TCT TIT TCA
TCA CCT CTT TCC ATT-3’ for deletion of residues 156170.
The deletion was performed on the cDNA encoding the mutant
[PV121,122RR]preproVIP, thereby resulting in a double mutant. The
introduced mutations were verified by sequence analysis. The mutated
cDNAs were subcloned as XbaI-EcoRI fragments into XbaI-EcoRI-
digested pBW91 resulting in pBW125 encoding PV121,122RR]prepro-
VIP and pBW135 encoding [PV121,122RR;d15617O]preproVIP.
2.2. Cell culture and transfection
NIH 3T3 cells from ATCC (MD, USA) were grown at 10% CO2 in
Dulbecco’s modified Eagles medium supplemented with 10% newborn
calf serum (NCS), 50 IU penicillin and 5O&ml streptomycin. The cells
were transfected with 20 fig of the peptide expression vector and
cotransfected with 2 pg of pSV2neo using the calcium phosphate pre-
cipitate procedure HO]. Stable clones were selected in NIH 3T3 medium
supplemented with 0.6 mg/ml G418 (Sigma).
24 h cell culture media were collected from a confluent 100 mm Petri
dish receiving fresh cell culture media containing only 0.5% newborn
calf serum 24 h before harvest.
Four different antisera recognizing different peptides in the VIP
precursor were used: antiserum Ab 5598 (the general VIP antiserum)
recognizing an internal region of VIP and reacting with both C- and
N-terminal extended VIP forms; antiserum Ab 5603 which is specific
for the amidated C-terminus of VIP, as it does not react with glycine-
extended VIP or VIP with a carboxy group at the C-terminus [ 11,121;
antiserum Ab 73 14 recognizing preproVIP 156170; and antiserum Ab
3668 recognizing both PHM and PHV [l] (see also Fig. 1 for the
recognition sites of the antisera).
2.4.1. Gelfiltration. 24 h cell culture media from stable clones were
aonlied to a Seohadex G 50 Sunerline column (11 x 1,000 mm), as
described . The columns were calibrated with synthetic VIP and
preproVIP 156 170. The fractions were freeze-dried and reconstituted
in assay buffer prior to radioimmunoassay. DB 2000 and vitamin B,,
were used as void volume marker and total volume marker, respec-
2.4.2. HPLC. The peak fractions of VIP immunoreactive material
in the gel filtration profiles of media from NIH- 9-l-E4 or NIH-20-2-
E2. were uooled (fractions 51-57 and 52-60 from the gel filtrations of
NIH-9-l-B4 and ‘NIH-20-2-E2, respectively). The pools were freeze-
dried and reconstituted in H,O with 0.1% TFA and analyzed by HPLC
as described previously [l]. The media from clone NIH-23-3-Cl were
applied directly to the column. Gradient elution was done at 30°C with
99% ethanol (Lichrosolv, Merck, Germany) containing 0.1% TFA (sol-
vent B). The elution gradient was isocratically 10% for 2 min, l&25%
solvent B for 4 min, 25-70% for 50 min, 70-80% for 4 min and isocrati-
tally for 10 min. The flow rate was 0.5 ml/min, and fractions of 0.5 ml
were collected, freeze-dried in a speed vacuum concentrator, reconsti-
tuted in assay buffer and analyzed by radioimmunoassay. The column
was calibrated in separate runs with synthetic VIP and preproVIP
2.4.3. cAMPassay. For CAMP assays a stable CHO clone, CHO-60-
l-A6 expressing the rat VIP receptor was used (the rat VIP receptor
cDNA was kindly provided by Prof. Nagata, Osaka Bioscience Insti-
tute, Osaka, Japan). 10’ cells/well were seeded in 24-well cell culture
dishes, and the following day the cells were washed twice in fresh NIH
3T3 cell culture media containing only 0.5% NCS, stimulated with 0.5,
5 or 50 ~1 24-h media from either NIH 3T3, NIH-9-l-E4 or NIH-23-
3,Cl cells in the presence of 0.1 mM I-methyl-3-isobutylxanthine.
cells were stimulated in 0.5 ml NIH 3T3 cell culture media containing
0.5% NCS for 45 min at 37°C. After the incubation the cells were
washed twice in Kreb’s-Ringer- HEPES buffer, pH 7.4, containing 2.5
mM CaCl,, 1 mM MgCl,, 0.1 g bovine serum albumin/100 ml, 1%
Bacitracin and 1 mM /I-mercaptoethanol.
incubating the cells for 20 rnin on ice in 100 ~1 0.4% perchloric acid.
After centrifugation, CAMP in the supematant was quantified using a
CAMP radioimmunoassay kit from Amersham (UK).
CAMP was extracted by
170lpreproVIP were established, and the preproVIP de-
rived peptides found in the media were characterized by
gel filtration combined with radioimmunoassays
for preproVIP 156-170 (Ab 7314), and VIP (Ab 5598).
Fractions from gel filtrations of the [PV 12 1,122RRJ 156
170lpreproVIP clones were also analyzed with the amide
specific VIP antiserum (Ab 5603). In addition, the VIP
immunoreactive material was characterized
combined with radioimmunoassay.
the gel filtrations were performed on 2-3 different clones,
and the HPLC characterizations
NIH 3T3 clones expressing
For each construct,
on at least two different
3.1. Processing of prepro VIP in NIH 3 T3 cells
Fig. 2 (upper panel) shows a gel filtration profile of the
peptides found in the media from a stable clone express-
ing preproVIP (NIH-9- 1 -E4). In the preproVIP 156170
profile, a part of the immunoreactive material eluted as
the synthetic standard. The VIP immunoreactivity
eluting earlier than the standard, indicating that VIP is
extended, probably at the C-terminus as this peak also
reacted with the radioimmunoassay
To further characterize the VIP immunoreactive mate-
rial eluting from the gel filtration column, the peak frac-
tions were pooled (peak I, fractions 51-57, Kd 0.36
0.52) and analyzed by HPLC (Fig. 2, lower panel). The
material eluted as a single peak reacting both with the
VIP and the preproVIP 156170-specific antisera, indi-
cating that this peak consists of C-terminally extended
VIP. No immunoreactive material for PHM or PHV (Ab
3668) could be detected (data not shown).
B.S. Wulff et al. IFEBS Letters 341 (1994) 43-48
Gly Lys Arg
153 154 155
GUY Lys Are
Processed VIP Precursor
Ab 5599 Ab 5603
Fig. 1. Schematic representation of preproVIP and the preproVIP derived peptides. The recognition sites of the antisera used are indicated.
3.2. Processing of [PVl21,122RR]prepro
The gel filtration profile of a stable clone expressing
[PV121,122RR]preproVIP is shown in Fig. 3 (upper
panel). The VIP immunoreactive material eluted as one
major peak at the same position as the wild-type peak
(Fig. 2). However, with the mutant no earlier eluting VIP
immunoreactivity was found, indicating that the intro-
duced tetrabasic cleavage site was completely cleaved. As
for the wild-type, a part of the preproVIP 156170 im-
munoreactive material eluted as the standard. The peak
(fractions 52-60, Kd 0.34-0.49) reacting with both VIP
(Ab 5598) and preproVIP 156-l 70-specific antisera were
pooled and characterized by HPLC (Fig. 3, lower panel).
The elution profiles were similar to the HPLC profiles
from the wild-type preproVIP (Fig. 2, lower panel).
VIP in NZH
3.3. Processing of[PVl21,122RR,Al56-170]preproVZP
in NZH 3T3 cells
In the gel filtration profiles from clones expressing this
mutant, VIP immunoreactive material eluted as the syn-
thetic standard. (Fig. 4, upper panel). Some of this mate-
rial also reacted with the amide-specific VIP antiserum,
demonstrating that a fraction of the VIP immunoreactive
material was amidated. As expected no preproVIP 156
170 immunoreactivity was found (data not shown).
To further characterize
found in the media, the media were applied to HPLC,
and the fractions were analyzed with the two VIP anti-
the VIP immunoreactivity
sera, Ab 5598 and Ab 5603 (Fig. 4, lower panel). The Ab
5598 immunoreactive material consisted of two peaks,
one co-eluting with the standard and also reacting with
the antiserum specific for amidated VIP, and a peak
eluting as a more hydrophilic peptide reacting only with
the Ab 5598 antiserum. This peak probably consists of
VIP C-terminally extended with the residues Gly-Lys-
3.4. Biological activity of the expressed peptides
The biological activity of the VIP immunoreactive ma-
terial produced by a clone expressing preproVIP (NIH-
9- 1 -E4) and by a clone expressing [pV12 1,122RRJ 156
17OlpreproVIP (NIH-23-3-Cl) was studied. The concen-
trations of immunoreactive material (both Ab 5598 and
Ab 5603) in the media from control cells, NIH-23-3,Cl
cells and NIH-9-l-E4 cells are shown in Table 1. The
control cells expressed no detectable amounts of either
Ab 5598 or Ab 5603 immunoreactivity.
produced comparable concentrations
munoreactive material (3.6 nM vs. 4.3 nM), whereas the
media from NIH-23-3,Cl cells contained approximately
a lOO-fold higher amount of Ab 5603 immunoreactivity
(specific for amidated VIP) than the NIH-9-l-E4
(2.7 nM vs. 23 PM).
Addition of 0.5 ~1 and 5 ,~l control media to CHO-60-
1 -A6 cells gave no measurable production of CAMP, and
addition of 50 ~1 control media resulted 1.4 pmol CAMP/
lo5 cells (Table 2). In contrast addition of 50 ,~l media
The two clones
of Ab 5598 im-
B.S. Wulff et al. IFEBS Letters 341 (1994) 43-48
156. 170 ,
Fig. 2. (Upper panel) Gel filtration profiles of VIP (Ab 5598, ?
preproVIP IS-170 (Ab 7314, Cl) immunoreativities in culture medium
from cells stably expressing preproVIP. (Lower panel) HPLC analysis
of the immunoreative VIP peak in the gel filtrations (fractions pooled
are indicated by open rectangle in upper panel). The fractions were
analyzed for VIP (Ab 5598) and preproVIP 156170 (Ab 7314) im-
munoreactivities. (symbols as above). The elution positions of VIP and
preproVIP 156-170 are indicated by open and closed arrows, respec-
from both clones to the CHO-60-l-A6 cells resulted in
a significant increase in CAMP (Table Z), and also 5 ~1
media from the two clones were able to increase the
CAMP production in CHO-60-1 -A6 cells.
When preproVIP is expressed in NIH 3T3 cells, very
low amounts of amidated VIP (Table 1) are found in the
cell culture media. This demonstrates, as also found for
other peptide precursors expressed in fibroblast cell lines
[ 14-161 that these cell lines have no or only limited proc-
essing capacity. Nevertheless,
munoreactive material is characterized by gel filtration,
one major peak is found. This peak also reacts with the
preproVIP 1X-170 antiserum but not with the PHMl
PHV antisera, indicating that the dibasic processing site
N-terminal to the VIP sequence is susceptible to some
kind of cleavage.
In an attempt to obtain expression of amidated VIP
in NIH 3T3 cells two preproVIP
pressed: (i) [PVl2 l,l22RR]preproVIP,
cleavage site N-te~inal to the VIP sequence was
changed into a tetrabasic
when the VIP im-
mutants were ex-
where the dibasic
cleavage site (Arg”‘,
0.2 0. 4
0. 6 0. 8
Fig. 3. (Upper panel) Gel filtration profiles of VIP (Ab 5598, e) and
preproVIP 156170 (Ab 7314, 0) immunoreativities in cell culture
medium from cells stably expressing
(Lower panel) HPLC analysis of the i~unoreactive
gel filtrations (fractions pooled are indicated by a rectangle in upper
panel). The fractions were analyzed for VIP (Ab 5598) and preproVIP
156170 (Ab 7314) immunoreactivities. The elution positions of VIP
and preproVIP 156-170 are indicated by open and closed arrows,
[pV12 1,l ZZRR]preproVIP.
VIP peak in the
B.S. Wulff et al. IFEBS Letters 341 (1994) 4348
0. 4 0. 6 0. 0 1. 0
20 40 60
Fig. 4. (Upper panel) Gel filtration profiles of VIP (Ab 5598, 0) and
amide specific VIP (Ab 5603,O) immunoreactivities in the cell culture
media from cells stably expressing [PV121,122RRJl56-170]prepro-
VIP. (Lower panel) HPLC analysis of VIP (Ab 5598) and amide-spe-
cific VIP (Ab 5603) immunoreactivities in the cell culture media.
Arg122,Lys123,Arg1ti), which fits the consensus sequence
Arg,X,Lys/Arg,Arg for cleavage
furin ; (ii)
preproVIP, a double mutant containing the tetrabasic
sequence and with residues 156170 deleted.
by the ubiquitous
VIP immunoreactive material in NIH 3T3, NIH-9-l-E4 and NIH-23-
?-Cl as determined by the general (Ab 5598) and the amide-specific VIP
Media from AB 5598 (PM)
Ab 5603 (OM)
*Below detection limit.
Induction of CAMP production by media from NIH 3T3, NIH-9-l-E4
and NIH-23-3-Cl cells
CAMP @M/lOS CAMP (pM/lO’ CAMP @M/10’
cells) produced cells) produced
after addition after addition
of 0.5 ~1 media of 5.0 ~1 media
(n = 4)
(n = 4)
mean f S.E.M. mean f S.E.M.
of 50 ~1 media
(n = 4)
mean f S.E.M.
1.0 f 0.1
1.1 f 0.2
5.2 f 2.6
12.6 f 3.3
1.4 * 0.3
23.5 f 0.5
36.5 k 3.4
The amount of CAMP produced after addition of, respectively 0.5, 5
or 50 ~1 media from the three cell lines to cell culture wells containing
CHO-60- 1 -A6 cells in 500 ~1 media was measured (< std, below detec-
The expression of [PV121,122RR]preproVIP
3T3 cells resulted in efficient cleavage at the cleavage site
N-terminal to VIP (Fig. 3 upper panel), but still no VIP
immunoreactive material eluting as the synthetic stand-
ard on gel filtration was found. The major part of the
VIP immunoreactive material
thetic standard on gel filtrations, and part of the im-
munoreactivity also reacted with the amide-specific VIP
antiserum (Fig. 4). HPLC analysis of the media showed
that the amide-specific immunoreactive
as the synthetic VIP standard. Consequently this cell line
contains both carboxypeptidase
dation enzyme. The expression of the amidation enzyme
mRNA in NIH 3T3 cells has previously been shown [ 181.
Although amidated VIP is expressed, the processing is
incomplete and the major part of the VIP immunoreac-
tivity detected with Ab 5598 elutes from the HPLC col-
umn as a more hydrophilic peptide. This indicates that
the peptide exists in an extended form, probably C-termi-
nally extended with Gly-Lys-Arg.
Nevertheless, we have shown that by introducing the
appropriate mutations in the peptide precursor, it is pos-
sible to synthesize amidated VIP in a non-endocrine cell
A minimum length of about 65 amino acids is required
for entering the secretory pathway [19,20], and for pro-
duction of small amidated or otherwise modified pep-
tides in non-endocrine cells, a C- or N-terminal extension
of the peptides are necessary. As also showed for the
protein C precursor , we found that it is possible to
alter the dibasic cleavage site in the VIP precursor in
such a way that the resulting precursor is efficiently
cleaved in non-endocrine cells. The same was found
when a tetrabasic mutant of human insulin was ex-
pressed in four different non-endocrine cell lines .
Besides, we could, by deleting the C-terminal peptide
(residues 156170), obtain amidation of the expressed
VIP. It has been suggested that expression of C-termi-
from cells expressing
eluted as the syn-
H-like activity and ami-
nally deleted peptide precursors could be used for the
expression of small amidated peptides in non-endocrine
cells . However, this concept could not be directly
applied to the expression of VIP as the C-terminally
truncated VIP is only partially processed N-terminal to
the VIP sequence. Furthermore
the signal peptide is probably less than the required
length to enter the secretory pathway , and conse-
quently a N-terminal extension of the peptide is required.
Efficient cleavage at this site was obtained by changing
the cleavage site N-terminal to VIP into a consensus
cleavage site for the ubiquitous processing enzyme furin.
Using this method it will be possible to express small
modified peptides, e.g. peptides with disulfide bridges,
amidated peptides and glycosylated peptides (e.g. endo-
thelin, calcitonin and vasopressin that all contain at least
one disulfide bridge), in non-endocrine cell lines, which
are generally used for large-scale production.
the processing in these cells can be optimized by engi-
neering the cell line to over-express
boxypeptidase E. Over-expression of furin in COS-7 cells
was found to increase the cleavage efficiency in a mu-
tated proinsulin with a tetrabasic cleavage site . An-
other possibility is to screen a number of non-endocrine
cell lines for the expression of furin, carboxypeptidase
and amidating enzyme, and choose a cell line with a
suitable enzyme combination.
Media from cells expressing [PV121,122RR,~l156
170lpreproVIP and from cells expressing preproVIP
were able to activate the VIP receptor as demonstrated
by induction of CAMP production in CHO cells express-
ing the VIP receptor. The highest amounts of CAMP was
produced after addition of 50 ~1 media from cells ex-
10’ cells). However, media from the cells expressing the
wild-type precursor also significantly
CAMP production, (50 ~1 media from preproVIP ex-
pressing cells produced 23 pmol CAMP/IO’ cells). This
indicates that VIP extended C-terminally by residues
15&170 is able to bind to and activate the receptor.
Although this extended VIP form is not found in tissue,
the observation corresponds
PHM and its C-terminally extended form, PHV has bio-
logical activity [ 131 and to the finding that both PACAP
27 and PACAP 38 are biologically active .
the length of VIP plus
furin and car-
to the finding that both
thanked for excellent technical assistance. The work was supported by
Professor Suad Al-Kassab’s Foundation, the Novo Foundation, the Ib
Henriksen Foundation, the Danish Foundation for the Advancement
Yvonne Ssndergard and Anita Hansen are
B.S. Wurff et al. IFEBS Letters 341 (1994) 4348
of Medical Science, The Gerda and Aage Haensch Foundation and the
Danish Biotechnology Center for Signal Peptide Research. Further-
more, the work was supported by the Danish Medical Research Coun-
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