Transglutaminase-mediated polyamination of vasoactive intestinal
peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity
Salvatore De Maria
1
, Salvatore Metafora
2
, Vittoria Metafora
2
, Francesco Morelli
2
,
Patrick Robberecht
3
, Magalı
`
Waelbroeck
3
, Paola Stiuso
4
, Alfredo De Rosa
1
, Anna Cozzolino
4
,
Carla Esposito
4
, Angelo Facchiano
5
and Maria Cartenı
`
1

(Spm), is reported. Appropriate binding assays and adeny-
late cyclase enzymatic determinations have shown that these
VIP adducts act as structural VIP agonists, both in vitro and
in vivo. In particular, their IC
50
and EC
50
values of human
and rat VIP/pituitary adenylate cyclase activating peptide
(PACAP)
1
and VIP/PACAP
2
receptors indicate that VIP
Dap
is a VIP agonist, with an affinity and a potency higher than
that of VIP, while VIP
Spd
and VIP
Spm
are also agonists but
with affinities lower than that of VIP. These findings suggest
that the difference in adduct agonist activity reflects the
differences in the positive charge and carbon chain length of
the polyamine covalently linked with the VIP Gln
16
residue.
In addition, the data obtained strongly suggest that the
length of polyamine carbon chain could be critical for the
interaction of the agonist with its receptor, even though

ligand binding and activation of G-protein coupled recep-
tors which use relatively small molecules as their ligands,
fewer results are available in the case of peptide receptors
which have ligands that are much larger and which exhibit
greater conformational flexibility. The detailed mechanism
of signal transduction mediated by the VIP receptor and the
physiological role of the different VIP receptors are
currently investigated. Furthermore, the only structural
information available on VIP has been mainly obtained by
CD and NMR analysis [10]. Recently, a conformational
study explored the theoretically preferred conformation of
VIP by combining experimental information with unre-
strained molecular calculation. The results of these studies
showed that (a): most VIP conformations, including the
global minimum, can be described as bent conformation; (b)
atype1bturn involves the residues of the VIP fragment
P2–5 and a different type of b-turn involves the residues of
the fragment P6–11; (c) the central portion (residues 7–15)
and the C-terminus (residues 19–27) are in a helical confor-
mation [11,12].
Little is known on the role played by the different VIP
residues in the recognition and activation of natural
receptors. Structural–activity studies, performed on a
Correspondence to S. Metafora, CNR International Institute of
Genetics and Biophysics, Via Pietro Castellino, 111-80131 Naples,
Italy. Fax: + 39 081 6132 253, Tel.: + 39 081 6132 254,
E-mail:
Abbreviations:MEM,minimalessentialmedium;CHO,Chinese
hamster ovary; Dap, 1,3-diaminopropane; iNOS, inducible nitric
oxide synthase; LPS, lipopolysaccharide; L-NAME, N

substitutions at this site did not affect the VIP affinity or
potency, suggesting that this region is not directly involved
in the recognition or activation of receptors. In contrast,
Robberecht et al. demonstrated the unexpected importance
of Gln16 in the central region of the secretin family peptides
for its interaction with the receptor N-terminal domain [18].
On the basis of this finding, we were prompted to use the
transglutaminase (TGase) to modify the primary structure
of VIP in order to investigate the effect of insertion at the
level of the Gln16 c-carboxyamide group of a variety of
amines of different carbon chain length and positive charge
on VPAC receptor activity, both in rats and humans [19–
23]. The functional characterization of three polyaminated
VIP derivatives demonstrated their ability to act as agonists
with an affinity and a potency higher than VIP (VIP
Dap
)or
with an affinity lower than VIP (VIP
Spd
and VIP
Spm
)on
VPAC receptors. The relevance of the polyamine carbon
chain length and positive charge on receptor activation has
been pointed out and the results of some experiments on
murine J774 macrophage cell cultures have shown the
ability of these VIP adducts to modulate in vivo the iNOS
activity at the level of transcription.
MATERIALS AND METHODS
VIP and Ro 25-1553 synthesis

M
Tris/HCl buffer, pH 8.0, containing 10 m
M
dithiothreitol, 2.5 m
M
CaCl
2
, and 0.2
M
Dap or Pt, or
Spd, or Spm, where required; 3 lg (6.7 mU) TGase were
added at the start of incubation, and the same amount of
enzyme was added after 6 h. A control sample incubated in
the absence of TGase was assayed simultaneously. At the
end of the incubation, the reaction mixtures were centrifuged
at 12 000 g for 10 min, and the resulting supernatants were
used to purify the VIP analogues by HPLC.
Purification and characterization of the VIP derivatives
The VIP analogues present in the supernatants were purified
by HPLC chromatography (Waters; Model 660 HPLC
apparatus) using an analytical reversed-phase Vydac C18
column (4.6 · 150 mm; Separations Group, Hesperia, CA).
The column was equilibrated with 0.01% trifluoroacetic
acid and elution was performed in 35 min (flow rate
1mLÆmin
)1
) at room temperature with a 0–60% aceto-
nitrile linear gradient. Fractions of 0.2 mL were collected
and the absorbance peaks were pooled and evaporated to
dryness. The dry samples were dissolved in distilled water

without geneticin.
Membrane preparation, receptor identification,
and adenylate cyclase determination
An appropriate number of CHO cells was harvested with
a cell scraper and pelleted by low speed centrifugation, the
supernatant was discarded and the sedimented cells were
lysed by addition of 1 m
M
NaHCO
3
and quick freezing in
liquid nitrogen. After thawing, the lysate was centrifuged
at 4 °C for 10 min at 400 g and the supernatant was
further centrifuged at 20 000 g atthesametemperature
andforthesametimelength.Thefinalpelletwas
resuspended in 1 m
M
NaHCO
3
and used immediately as a
crude membrane preparation. [
125
I]VIP (specific radio-
activity, 0.7 mCiÆmmol
)1
) was used as tracer for the
identification of both rat or human VPAC
1
receptors;
[

bound radioactivity was separated from the free radioac-
tivity by filtration through glass fibre filters GF/C
presoaked for 24 h in 0.1% polyethyleneimine and rinsed
three times with a 20 m
M
phosphate buffer (pH 7.4)
containing 1% BSA. Adenylate cyclase activity was
determined by a previously published technique [29].
Membrane proteins (3–15 lg)wereincubatedinatotal
volume of 60 lL containing 0.5 m
M
[a-
32
P]ATP, 10 l
M
GTP, 5 m
M
MgCl
2
, 0.5 m
M
EGTA, 1 m
M
cAMP, 1 m
M
theophylline, 10 m
M
phosphoenolpyruvate, 30 lgÆmL
)1
pyruvate kinase, and 30 m

days. For use, cells were seeded into 12-well plates (Falcon)
and allowed to adhere for 2 h. After this, medium was
replaced with fresh medium containing either 0.01 lgÆmL
)1
lipopolysaccharide (LPS; this complex molecule is a com-
ponent of the Gram-negative bacteria outer membrane
possessing a strong iNOS-inducing activity on murine
macrophages) alone (control), or VIP and its polyaminated
adducts (10
)10
)10
)6
M
), alone or in combination with LPS,
and the cells were incubated at 37 °C for a further 24 h in an
humidified atmosphere containing 5% CO
2
and 95% air.
Cell viability was measured by both Trypan blue exclusion
test and MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; Sigma Aldrich]. In specific control
inhibition experiments dexamethasone (10
)6
M
;Sigma)was
added to macrophages treated with either 0.01 lgÆmL
)1
LPS alone, or VIP and its polyaminated adducts
(10
)10

)10
)10
)6
M
), alone or in combination with LPS. After
the end of the incubation time, the cells were rinsed three
times with ice-cold NaCl/Pi, removed from the culture
plates with a cell scraper, collected, and transferred to
microcentrifuge tubes. The sedimented cells were lysed by
addition of 50 lL ice-cold hypotonic homogenization
buffer (1 m
M
EDTA, 1 m
M
hypotonic EGTA, 25 m
M
Tris/HCl pH 7.4). The iNOS activity occurring in 20 lg
of homogenate proteins was evaluated by a NOS Detection
Assay Kit (Stratagene) [31] according to the manufacturer’s
instructions. In this assay, [
3
H]arginine (50 CiÆmmol
)1
;
Amersham) was used as substrate and the reaction mixture
was incubated for 30 min at 37 °C. Two blanks were
included in the assay: one was prepared by omitting the
homogenate, the other by adding the iNOS inhibitor
N
x

(Superscript II; Life Technologies) at 37 °Cfor1.5h
according to the manufacturer’s protocol (final volume
20 lL). The cDNA contained in 2 lL of this reaction
mixture was amplified in another reaction mixture con-
taining, in a final volume of 25 lL, 10 m
M
Tris/HCl
pH 8.3, 1.5 m
M
MgCl
2
, 50 m
M
KCl, 100 ng of both sense
and antisense primers for iNOS (sense, 5¢-GTTTCT
TGTGGCAGCAGC-3¢;antisense,5¢-CCTCGTGGCT
TTGGGCTCCT-3¢), 100 l
M
deoxynucleoside triphos-
phate, and 1 U Taq DNA polymerase (Roche Diagnos-
tics). The reaction was carried out in a DNA thermal
cycler (Promega). The PCRs were performed with 35
cycles in the exponential phase of amplification and always
started with a 3-min denaturation step at 95 °Cand
terminated with a final 7 min step at 72 °C. The cycle for
iNOS was 95 °C, 45 s; 56 °C, 45 s; 72 °C, 45 s. The PCR
products were analysed by electrophoresis on a 1.2%
agarose gel in Tris/borate/EDTA [32]. The identities of the
amplification products were confirmed by comparison of
their sizes with the sizes expected from the known gene

pig), VIPR_RAT
(VPAC
1
rat), VIPS_HUMAN (VPAC
2
human), VIPS_
MOUSE (VPAC
2
mouse), VIPS_RAT (VPAC
2
rat). The
Fig.1.EffectofVPAC
1
and VPAC
2
ligands (VIP and its polyaminated agonists) on membrane binding and adenylate cyclase activity. The data
reported in the figure refer to: (1) Dose-dependent inhibition of
125
I-labelled ligand ([
125
I]VIP was used for the identification of rat or human VPAC
1
receptors, whereas [
125
I]Ro 25-1553 was used for labelling of rat or human VPAC
2
receptors) binding (panels A, C, E, and G) to crude preparations
of CHO cell membranes expressing recombinant VPAC
1
and VPAC

was carried out by considering the domain assignment
reported in the SwissProt database.
Statistical analysis
The data have been reported as means ± SEM of at least
three different determinations. The means were compared
using analysis of variance (one-way
ANOVA
)plusBonfer-
roni’s t-test and a P-value < 0.05 was considered significant.
The software packages used for statistical analysis were
GRAPHPAD INSTAT
and
MINITAB
. The curve fitting programs
used were in
GRAPHPAD PRISM
,
GRAPHPAD INPLOT
, and
MINITAB
.
RESULTS
A positively charged amino acid into position 16
modulates the VIP ability to bind its specific receptors
VIP and its three polyaminated adducts (VIP
Dap
, VIP
Spd
,
and VIP

and human VPAC
1
receptors [33], could allow a better
identification of the polypeptide regions involved in the
ligand/receptor molecular interactions.
The polyaminated VIP adducts are agonists of either
higher or lower affinity and potency than VIP
The effect of the three polyaminated VIP adducts on the
human and rat VPAC
1
and VIPAC
2
receptor activity was
evaluated by evaluating the adenylate cyclase enzymatic
activity of a crude preparation of membranes. The data
reported in Fig. 1 (panels B, D, F, H) and Table 1 indicate
that VIP
Dap
has a higher apparent affinity and higher
maximum effect than VIP in all the receptors tested. In
contrast, VIP
Spd
and VIP
Spm
werefoundtoactwithalower
apparent affinity, their EC
50
values being 3–10 times higher
than the VIP value (Table 1). The data on the relative
potencies of Spd- and Spm-conjugated VIP in cAMP

M
) required for 50% tracer binding inhibition; EC
50
, peptide concentration (n
M
) required for half maximal stimulation of adenylate cyclase activity;
IA, intrinsic activity, the ratio between the maximal stimulating effect of modified VIP and that of VIP. *P <0.05,**P < 0.01 (Bonferroni’s t-test) vs. the VIP value.
Human Receptor Rat Receptor
VPAC
1
VPAC
2
VPAC
1
VPAC
2
Ligand IC
50
EC
50
IA IC
50
EC
50
IA IC
50
EC
50
IA IC
50

with this result, we now report data that demonstrate the
ability of the VIP/LPS combination to modulate the
capacity of J774 macrophages to generate NO in a biphasic
manner, the lower concentrations of VIP being more active
(a maximum stimulation was reached at 10
)8
M
) than the
higher concentrations (10
)6
M
) (Fig. 2, upper panel). Sim-
ilar results were obtained with equimolar concentrations of
polyaminated VIP adducts, the VIP
Dap
adduct being the
most active. The NO production profile obtained either with
VIP or polyaminated VIP adducts was similar to the iNOS
activity profile induced by the same molecules (Fig. 2, upper
and lower panels). In turn, the increase of iNOS activity
produced by VIP or its polyaminated adducts was associ-
ated with a marked increase in the expression of the gene
encoding iNOS, as evaluated by semiquantitative RT/PCR
(Fig. 3).
DISCUSSION
In this report we have shown that polyamination of Gln16
side chain significantly modulates the ability of VIP to bind
and stimulate the VPAC
1
receptor. This finding supports

was incubated for 30 min at 37 °C. iNOS activity was expressed as
citrulline pmolÆmg protein
)1
Æmin
)1
. Controls: cells untreated (unfilled
bars) or treated with LPS alone (0.01 lgÆmL
)1
; diagonal bars, \).
Experimental: cells treated with LPS (0.01 lgÆmL
)1
) in combination
with different concentrations of VIP (cross hatched bars, X) or VIP
agonists (VIP
Dap
, speckled bars; VIP
Spd
, diagonal bars, /) VIP
Spm
,
horizontal bars). Further experimental details are reported in
Materials and methods.
Fig. 3. Expression of the gene encoding iNOS in J774 macrophages
following their treatment with various VPAC
1
and VPAC
2
ligands (VIP
and its agonists VIP
Dap

arose gels. Further experimental details are reported in Materials and
methods.
3216 S. De Maria et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Robberecht’s data indicating the critical role played by the
presence of a positively charged amino acid (arginine, R) at
position 16 of VIP polypeptide chain [18]. In addition, the
possibility that the side chain length could play an important
role in modulating the receptor recognition ability and
activity is supported by our IC
50
and EC
50
data that show
the best performance of VIP
Dap
activity in comparison with
VIP, VIP
Spd
, and VIP
Spm
. The high affinity of VIP
Dap
might
be related to specific interactions of this agonist with
well-defined hydrophilic regions of the receptor polypeptide
chain. However, the possibility that additional hydrophobic
contact made by the R side-chain or by Dap may
significantly contribute to the binding affinity of these
agonists, cannot be ruled out on the basis of our present
data. The possible existence in the receptor of different

region/s involved in the interaction with VIP
Dap
, but also to
define the type of receptor–ligand interaction triggered by
the ligand binding process. Novel chemical modifications of
a peptide ligand, similar to those reported in this paper and
capable of both modulating the receptor activity and
increasing the discrimination capacity between receptor
subclasses, could be of the highest interest for a better
control of definite biological functions. It is also interesting
to note that these chemical modifications at the site 16
associated with appropriate modifications at the level of
other residues in the VIP N terminus could be useful for the
production of better VIP antagonists. In addition, the
substitution of Arg16 in R-VIP [18] with a polyaminated
derivative of glutamine (Dap) could make the modified
peptide not only a better agonist or antagonist but also
protect its structural integrity from a trypsin-like proteolytic
attack.
The hypothesis that a positive charge at the position 16 of
VIP polypeptide chain could play an important role in the
ligand–receptor recognition mechanism is also supported by
the published data on charge distribution in VPAC receptor
amino acid sequences (see Fig. 4). In fact, these data show
the occurrence in the extracellular domains of VPAC
receptors of a significant clustering of well conserved
negative charges that could originate electrostatic interac-
tions with the positive charge(s) present at position 16 of
VIP adducts. Moreover, differential negative charge distri-
bution in the N-terminal domain and first extracellular loop

receptors and different VIP and LPS concentrations to
measure the effect of VIP and other substances on an LPS-
activated cell system [36]. The inhibitory effect observed at
high concentrations of VIP and its polyaminated adducts is
probably related to either the negative regulatory effect
exerted by the relatively high levels of VIP (NF-jB inhibition
by a cAMP-independent pathway [39]): or to a shedding
process of membrane-bound CD14 receptors from LPS-
stimulated macrophages induced by the highest concentra-
tions of VIP or its adducts used in our experiments [40] or
both. Experiments are in progress to elucidate the molecular
mechanism at the basis of the up-regulatory effect of these
ligands on iNOS gene expression. Finally, the obvious
discrepancies between biological activity (evaluated in vivo
on J774 macrophage cell line) and receptor binding affinity
(assessed in vitro on CHO cell-derived crude membranes) of
the various VIP derivatives (compare Fig. 1 with Fig. 2)
might be related to the different experimental conditions in
which these parameters were evaluated and to possible
differences between CHO cells and J774 macrophages in
membrane signal transduction mechanisms.
ACKNOWLEDGEMENTS
We thank P. De Neef, J. Cnudde, S. Baiano and F. Moscatiello for their
skilful technical assistance. This research was supported by a Grant
from ÔProgramma di Intervento per la Promozione della Ricerca
Scientifica in Campania L.R. n.41-31/12/94Õ.
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