Báo cáo Y học: Transglutaminase-mediated polyamination of vasoactive intestinal peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity

Transglutaminase-mediated polyamination of vasoactive intestinal

peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity

Salvatore De Maria

, Salvatore Metafora

, Vittoria Metafora

, Francesco Morelli

Patrick Robberecht

, Magalı

`Waelbroeck

, Paola Stiuso

, Alfredo De Rosa

, Anna Cozzolino

Carla Esposito

, Angelo Facchiano

and Maria Cartenı

Department of Experimental Medicine and Centro di Ricerca Interdipartimentale di Scienze Computazionali e Biotecnologiche, II

University of Naples, Italy;

CNR Institute of Genetics and Biophysics Adriano Buzzati Traverso, Naples, Italy;

Department of

Biochemistry and Nutrition, Medical School of Medicine, Universite

´Libre de Bruxelles, Bruxelles, Belgium;

Department of

Chemistry, University of Salerno, Salerno, Italy;

Istituto di Scienze dell¢Alimentazione, CNR, Avellino, Italy

Previous data showing an increase of receptor binding

activity of [R16]VIP, a vasoactive intestinal peptide (VIP)

structural analogue containing arginine at the position 16 of

its amino acid sequence, have pointed out the importance of

a positive charge at this site. Here, the functional charac-

terization of three VIP polyaminated adducts (VIP

Dap

VIP

Spd

, and VIP

Spm

), obtained by a transglutaminase-

catalysed reaction between the VIP Gln16 residue and

1,3-diaminopropane (Dap), spermidine (Spd), or spermine

(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

and EC

values of human

and rat VIP/pituitary adenylate cyclase activating peptide

(PACAP)

and VIP/PACAP

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

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

possible hydrophobic interaction cannot be ruled out. In vivo

experiments on murine J774 macrophage cell cultures have

shown the ability of these compounds to stimulate the

inducible nitric oxide synthase activity at the transcriptional

level.

Keywords: NO/iNOS; polyamines; transglutaminase; VIP

agonists; VIP receptors.

Vasoactive intestinal polypeptide (VIP) is a 28-amino acid

long peptide that serves the function of hormone, neuro-

transmitter, and immuno-modulator in mammals and other

vertebrates. It belongs to the important family of brain/gut

hormones including secretin, glucagon, pituitary adenylate

cyclase activating peptide (PACAP), etc. [1–3]. Although

originally identiﬁed on the basis of its strong vasodilating

activity, VIP exerts a wide spectrum of biological effects on

a number of target organs mediated by its interaction with

two distinct G-protein coupled receptors (VIP/PACAP

and VIP/PACAP

or VPAC

and VPAC

), which transduce

the ligand signal through the activation of different

enzymatic effector systems, such as adenylate cyclase,

phospholipase C, and inducible nitric oxide synthase (iNOS)

[4–9].

While work is more advanced on the mechanism of

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 ahelical 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: metafora@iigbna.iigb.na.cnr.it

Abbreviations:MEM,minimalessentialmedium;CHO,Chinese

hamster ovary; Dap, 1,3-diaminopropane; iNOS, inducible nitric

oxide synthase; LPS, lipopolysaccharide; L-NAME, N

-nitro-

-arginine methyl ester; NO, nitric oxide; PACAP, pituitary adenylate

cyclase activating peptide; Pt, putrescine; Spd, spermidine; Spm,

spermine; TGase, transglutaminase; VIP, vasoactive intestinal pep-

tide; VPAC

, VIP/PACAP

receptor; VPAC

, VIP/PACAP

receptor.

(Received 11 February 2002, revised 14 May 2002,

accepted 15 May 2002)

Eur. J. Biochem. 269, 3211–3219 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.02996.x

number of analogues and different VIP fragments, demon-

strated that full action of VIP is critically dependent on the

integrity of the entire molecule [13]. The VIP N-terminal

helix is known to be critical for the high afﬁnity binding and

coupling to the effector system, while the C-terminal

sequence has been shown to be important for VPAC

and

VPAC

discrimination [14–17]. Concerning the central

region of the VIP polypeptide chain, different amino acid

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

These peptides were synthesized as C-terminal amides by

solid phase methodology on an automated Applied

Biosystem apparatus using Fmoc chemistry as described

previously [24]. The peptides were cleaved and puriﬁed by

RP-HPLC on an apparatus using a DBV 300A (10 ·1cm)

column and by ion exchange chromatography on a Mono S

HR 5/5 column. Peptide purity (95%) was assessed by

capillary electrophoresis and sequence conformity was

veriﬁed by direct sequencing and ion spray MS.

TGase-catalysed synthesis of VIP derivatives

TGase activity was preliminarily assayed by determining the

-dependent covalent binding of amines to the VIP

peptide acting as amino acceptor substrate. Analysis of the

reaction products was performed by SDS/PAGE, followed

by ﬂuorography [25,26], using radioactive putrescine (Pt),

spermidine (Spd) or spermine (Spm) as amino donor

substrates.

Each preparation of c-(glutamyl16)-Dap-VIP (VIP

Dap

c-(glutamyl16)-Spd-VIP (VIP

Spd

), and c-(glutamyl16)-Spm-

VIP (VIP

Spm

) was obtained by incubating for 12 h in a ﬁnal

volume of 200 lLat37C50lgofnativeVIPwithTGase

in 125 m

Tris/HCl buffer, pH 8.0, containing 10 m

dithiothreitol, 2.5 m

CaCl

, and 0.2

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 gfor 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

) 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

and submitted to ES-MS, as described previously [27].

CHO cell line culture

The recombinant Chinese hamster ovary (CHO) cells

expressing the rat or human recombinant VPAC

and

VPAC

receptors were prepared in P. Robberecht’s labor-

atory (Department of Biochemistry and Nutrition, Medical

School of Medicine, Universite

´Libre de Bruxelles, Bel-

gium). Cells were incubated at 37 Cina-minimal essential

medium (a-MEM), supplemented with 10% fetal bovine

serum, 2 m

-glutamine, 100 lgÆmL

penicillin and

100 lgÆmL

streptomycin, with an atmosphere of 95%

air and 5% CO

. Geneticin (0.4 mgÆmL

) was always

present in the culture medium of stock cultures. Subcultures

used for membrane puriﬁcation were grown in a medium

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

NaHCO

and quick freezing in

liquid nitrogen. After thawing, the lysate was centrifuged

at 4 C for 10 min at 400 gand the supernatant was

further centrifuged at 20 000 gatthesametemperature

andforthesametimelength.Theﬁnalpelletwas

resuspended in 1 m

NaHCO

and used immediately as a

crude membrane preparation. [

125

I]VIP (speciﬁc radio-

activity, 0.7 mCiÆmmol

) was used as tracer for the

identiﬁcation of both rat or human VPAC

receptors;

[

125

I]Ro 25–1553 (speciﬁc radioactivity, 0.8 mCiÆmmol

)

was used as tracer for labelling the rat or human VPAC2

receptors [28]. The binding of labelled ligands to puriﬁed

CHO membranes was performed as described [14]; in all

cases the nonspeciﬁc binding was deﬁned as the residual

binding in the presence of 1 l

VIP. Competition curves

were carried out by incubating membranes and tracer in

the presence of increasing concentrations of unlabelled

peptides. Peptide potency was expressed as IC

value, i.e.

as the peptide concentration required for half maximal

inhibition of tracer binding. In detail, the binding was

performed at 37 C in a buffer containing 20 m

Tris/

maleate pH 7.4, 2 m

MgCl

, 0.1 mgÆmL

bacitracin,

3212 S. De Maria et al. (Eur. J. Biochem. 269)FEBS 2002

and 1% BSA; 3–30 lg protein was used per assay. The

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

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

[a-

P]ATP, 10 l

GTP, 5 m

MgCl

, 0.5 m

EGTA, 1 m

cAMP, 1 m

theophylline, 10 m

phosphoenolpyruvate, 30 lgÆmL

pyruvate kinase, and 30 m

Tris/HCl at a ﬁnal pH of

7.5. The reaction was initiated by membrane addition and

was terminated after a 12-min incubation at 37 Cby

adding 0.5 mL of stop buffer (0.5% SDS, 0.5 m

ATP,

0.5 m

cAMP, 20 000 c.p.m. [8

H] cAMP). cAMP was

separated from ATP by two successive chromatographies

on Dowex 50-WX8 and neutral alumina.

Macrophage cell culture

The murine monocyte/macrophage cell line J774 (ATCC

TIB 67) was grown as monolayers in tissue-culture ﬂasks

(75 cm

growth area; Falcon) in Dulbecco’s MEM sup-

plemented with 10% (v/v) fetal bovine serum (Euroclone,

UK), 4 m

-glutamine, 100 unitsÆmL

penicillin, and

100 lgÆmL

streptomycin (standard culture medium). Cells

were harvested by gentle scraping and passaged every 3–6

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

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

), 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

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

;Sigma)was

added to macrophages treated with either 0.01 lgÆmL

LPS alone, or VIP and its polyaminated adducts

(10

)10

), alone or in combination with LPS.

Nitric oxide measurement

The NO produced by the iNOS-catalysed reaction was

evaluated by measuring with the Griess reagent nitrite

released by the macrophages into the culture medium [30].

Following 24 h incubation at 37 C, 400-lL aliquots of

culture medium were taken from the plates containing the

cell monolayers, mixed with an equal volume of Griess

reagent (0.5% sulfanilamide and 0.05% N¢-1-naphtylethy-

lenediamine dihydrochloride in 2.5% phosphoric acid), and

incubated at room temperature for 10 min The absorbance

of the coloured solution was measured at 570 nm. The

amount of nitrites released into culture medium was

expressed as nmol nitrites per 5 ·10

cells per 24 h, using

a sodium nitrite curve as a standard. Control experiments

demonstrated that VIP and VIP adducts did not interfere

with the Griess reaction.

Evaluation of iNOS activity

iNOS activity was determined in crude homogenates of

J774 cells. An appropriate number of cells was incubated

for 24 h in the absence or presence of either LPS

(0.01 lgÆmL

) or VIP or its polyaminated adducts

(10

)10

), 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

EDTA, 1 m

hypotonic EGTA, 25 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, [

H]arginine (50 CiÆmmol

;

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

-nitro-

-arginine methyl ester (

-NAME; 1 m

)tothe

reaction mixture before the homogenate. The iNOS

activity was expressed as citrulline pmolÆmg

protein

Æmin

. Control experiments demonstrated that

VIP and polyamines (Dap, Spd, Spm) did not interfere

with the iNOS activity.

RT-PCR

Messenger RNA, isolated by the mRNA Capture Kit

(Roche Diagnostics) from the J774 macrophages incuba-

ted in the standard culture medium for 24 h in the

presence of either 0.01 lgÆmL

LPS, or VIP and its

polyaminated adducts (10

)10

), alone or in combi-

nation with LPS, was transcribed by reverse transcriptase

(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

Tris/HCl

pH 8.3, 1.5 m

MgCl

, 50 m

KCl, 100 ng of both sense

and antisense primers for iNOS (sense, 5¢-GTTTCT

TGTGGCAGCAGC-3¢;antisense,5¢-CCTCGTGGCT

TTGGGCTCCT-3¢), 100 l

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

sequence. Coampliﬁcation of a different cDNA sequence

was performed by adding into the ampliﬁcation reaction

mixture the b-actin gene primers (10 ng of both sense and

antisense primers: sense, 5¢-CGTGGGCCGCCCTAGG

CACCA-3¢;antisense,5¢-TTGGCCTTAGGGTTCA

GGGGGG-3¢). No products were detectable in control

ampliﬁcations performed in the absence of cDNA (data

FEBS 2002 VIP polyaminated agonists and receptor activity (Eur. J. Biochem. 269) 3213

not shown). The semiquantitative evaluation of the PCR

products was achieved by integrating the peak area

obtained by densitometry of the ethidium bromide stained

agarose gels [software used: NIH image V.16; iNOS

(600 bp): 109, 757, 3300, 4581, 1159, 901; b-actin (300 bp):

670, 797, 833, 963, 819, 831]. The ratio between the yield

of each ampliﬁed product and coampliﬁed b-actin (iNOS/

b-actin mRNA ratio: 0.162, 0.949, 3.961, 4.757, 1.415,

1.084) allows a relative estimate of mRNA levels in the

samples analysed.

Multiple alignment and charge distribution in receptor

sequences

The amino acid sequences of the VIP receptors analysed

were derived from the SwissProt database. The following

sequences were used for the multiple alignment analysis:

VIPR_CARAU (VPAC

goldﬁsh), VIPR_HUMAN

(VPAC

human), VIPR_PIG (VPAC

pig), VIPR_RAT

(VPAC

rat), VIPS_HUMAN (VPAC

human), VIPS_

MOUSE (VPAC

mouse), VIPS_RAT (VPAC

rat). The

Fig.1. EﬀectofVPAC

and VPAC

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

receptors, whereas [

125

I]Ro 25-1553 was used for labelling of rat or human VPAC

receptors) binding (panels A, C, E, and G) to crude preparations

of CHO cell membranes expressing recombinant VPAC

and VPAC

receptors, by VIP (s), VIP

Dap

(d),VIP

Spd

(h), and VIP

Spm

(j); the results

are the means of three diﬀerent determinations and are expressed as the percentage of tracer speciﬁcally bound; (2) Dose-eﬀect curves of VIP (s),

VIP

Dap

(d) on adenylate cyclase activation (B, D, F, and H) in crude preparations of membranes from CHO cells expressing recombinant VPAC

and VPAC

receptors; the results, expressed in percentage increase of

P-labelled cyclic AMP produced in the presence of 1 l

VIP, are the means

of three diﬀerent experiments. The cAMPase activity was evaluated by a previously published radiometric assay [29]. Further experimental details

are reported in Materials and methods.

3214 S. De Maria et al. (Eur. J. Biochem. 269)FEBS 2002

multiple alignment was created by the

CLUSTALW

software.

Colours were added manually by considering the common

colour-code for charged amino acids (i.e. red for acidic, and

cyan/blue for basic side chains). The analysis of charge

distribution in the extracellular and cytoplasmatic domains

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

Spm

) possessing a different positively charged side

chain at position 16, were ﬁrst characterized by appropriate

binding experiments to VPAC receptors, both in humans

and rats. The data reported in Fig. 1 (panels A, C, E, G)

and analysed in Table 1 demonstrate that the VIP

Dap

adduct has a higher afﬁnity (lower IC

value) than VIP on

both rat and human VPAC

receptors, and a similar

afﬁnity to VIP on VPAC

receptors. The VIP

Spd

and

VIP

Spm

derivatives were 30–100-fold less potent than VIP.

The effect of the agonists used in these experiments was

tested on VPAC

and VPAC

receptors in both rat and

human on the assumption that the analysis of the data

obtained, associated with the knowledge of the structural

differences between these two receptors and between the rat

and human VPAC

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

and VIPAC

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

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

generation assays (not shown in Fig. 1) indicate that the

decrease in biological activity of these adducts reﬂects the

apparent decrease in their binding afﬁnity at the lowest

concentrations used (10

)10

), even though at the

highest concentrations (10

)10

) the biological activity

improves signiﬁcantly. By comparing the IC

and EC

Table 1. IC

and EC

values (n

) from binding experiments and adenylate cyclase assays. Experimental details are described in Materials and methods. Values are means ± SEM and are the means of at least

three diﬀerent determinations. IC

, Peptide concentration (n

) required for 50% tracer binding inhibition; EC

, peptide concentration (n

) 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

Ligand IC

IA IC