A novel Schistosoma mansoni G protein-coupled receptor is responsive to histamine.

Abstract

A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR) shared about the same level of sequence homology (approximately 30%) with all major types of amine GPCRs and could not be identified on the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual asparagine substitution (Asn(111)) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology, the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was responsive to histamine, which caused a dose-dependent elevation in intracellular Ca2+ (EC50=0.54+/-0.05 microM). An Asn(111)-->Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor activity, using assays that measured both Ca2+- and cAMP-mediated signaling. The results suggest that SmGPCR is a novel structural class of histamine receptor that may be unique to flatworms.

Full text

Page 1

Molecular & Biochemical Parasitology 119 (2002) 75–86
A novel Schistosoma mansoni G protein-coupled receptor is
responsive to histamine?
Fadi F. Hamdana,1, Mark Abramovitzb, Aisha Mousaa, Jinling Xiea,
Yves Durocherc, Paula Ribeiroa,*
aInstitute of Parasitology, McGill Uni?ersity, 21,111 Lakeshore Road, Ste. Anne de Belle?ue, Que., Canada H9X 3V9
bDepartment of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, PO Box 1005, Pointe Claire, Dor?al,
Que., Canada H9R 4P8
cNRC Biotechnology Research Institute, 6100 Royalmount A?enue, Montreal, Que., Canada H4P 2R2
Received 8 August 2001; received in revised form 1 October 2001; accepted 2 October 2001
Abstract
A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural
characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR)
shared about the same level of sequence homology (?30%) with all major types of amine GPCRs and could not be identified on
the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual
asparagine substitution (Asn111) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA
was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in
cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes
demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology,
the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was
responsive to histamine, which caused a dose-dependent elevation in intracellular Ca2+(EC50=0.54?0.05 ?M). An Asn111?
Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for
agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor
activity, using assays that measured both Ca2+- and cAMP-mediated signaling. The results suggest that SmGPCR is a novel
structural class of histamine receptor that may be unique to flatworms. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Schistosoma mansoni; G protein-coupled receptor; Biogenic amine; Histamine; Cloning; Neurotransmitter; Signal transduction
www.parasitology-online.com.
1. Introduction
Parasitic platyhelminths (flatworms) of the genus
Schistosoma are among the most prevalent causes of
human parasitic infection and disease. Schistosoma
mansoni is a causative agent of schistosomiasis, a debil-
itating disease that afflicts millions of people world-
wide. Studies of flatworm neurobiology have identified
several putative neurotransmitters, among them bio-
genic monoamines, which are present within the central
nervous system and peripheral tissues of the parasite.
The monoamine, serotonin (5-hydroxytryptamine: 5-
HT)), has been most extensively studied, and its many
functions as a modulator of motility and metabolic
regulator have been well documented (reviewed in [1]).
In contrast, considerably less is known about the prop-
erties of other biogenic amines, in particular histamine.
Although better known as a mediator of immunity,
histamine also functions as an important neurotrans-
mitter in a variety of vertebrate and invertebrate phyla
(see [2–4]). Among the flatworms, histamine has been
identified in S. mansoni [5] and other trematodes [6], as
?Note: Nucleotide Sequence data reported in this paper are avail-
able in the GenBank™ data base (SmGPCR: accession number:
AF031196).
* Corresponding author. Tel.: +1-514-398-7607; fax: +1-514-398-
7857.
E-mail address: paula–ribeiro@maclan.mcgill.ca (P. Ribeiro).
1Present address: Laboratory of Bioorganic Chemistry, Bldg. 8A,
NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
0166-6851/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S0166-6851(01)00400-5

Page 2

F.F. Hamdan et al. / Molecular & Biochemical Parasitology 119 (2002) 75–8676
well as several tapeworms (cestodes) such as Hymenolepis
diminuta and Diphyllobothium dendriticum [7,8]. In situ
immunofluorescence studies of D. dendriticum and the
frog trematode, Haplometra cylindracea identified his-
tamine-immunoreactive neurons within the cerebral gan-
glia and nerve cords of the central nervous system [6,8],
suggesting that histaminergic innervation is conserved in
this group of organisms. Histamine can be metabolized
in flatworms [7], but it is unknown if it is synthesized
endogenously and/or taken up directly from the host
[6,7]. Studies on the function of histamine reported an
effect on motor activity both in S. mansoni [5] and H.
diminuta [9], suggesting a role in neuromuscular trans-
mission or modulation.
To date, studies of histamine receptors have been
restricted largely to mammalian systems. A total of four
structural classes of histamine (H) receptors (named
H1–H4) have been identified in mammals, all of which
belong to the superfamily of G protein-coupled receptors
(GPCR). H1, H2 and H3 share little sequence homology,
typically less than 30%, and differ on the basis of binding
profiles and signaling mechanisms (see [10] for review).
H1 has characteristically high affinity for histamine (Kd
in the low nM range) and signals preferentially by
coupling to members of the Gq/11 protein family,
causing elevation in intracellular inositol phosphates and
mobilization of Ca2+. In contrast, the H2 receptor has
low affinity for histamine (Kd in the ?M range) and
couples primarily to a Gs protein, causing activation of
adenylate cyclase and an increase in intracellular cAMP
[10]. H3 is structurally related to the more recently cloned
H4 receptor [11,12], both of which have high affinity for
histamine (nM Kd) and signal through a common
Gi/o-mediated inhibition of adenylate cyclase. Alterna-
tive splicing of the H3 primary transcript yields multiple
isoforms with different functional properties and distri-
bution patterns [13], which further increases the reper-
toire of histamine receptors available in mammalian
systems. By comparison, very little is known about the
mode of action of histamine among invertebrates. No
histamine receptor has yet been cloned from any inver-
tebrate, even though histamine is known to be biologi-
cally active in most invertebrate phyla [4]. A search of
the complete genome of Caenorhabditis elegans, a free-
living nematode, and Drosophila failed to identify any
clear homologues of mammalian histamine receptors,
suggesting that invertebrate histaminergic GPCRs are
structurally distinctive.
In this study, we describe a first invertebrate histamine
receptor, a novel S. mansoni cDNA (SmGPCR), which
has structural characteristics of the amine GPCR family
but does not clearly resemble any of the mammalian
histamine receptors. Histamine activation of SmGPCR
in a heterologous system triggered mobilization of intra-
cellular calcium but not cAMP, suggesting the new S.
mansoni histamine receptor may be linked to Gq/11
proteins. Several unusual features of this receptor, in-
cludingasurprisingAsp?Asn
transmembrane (TM) domain 3, point to SmGPCR as
a new structural class of histamine receptor, which
diverged early in evolution and may be unique to
flatworms.
substitution in
2. Materials and methods
2.1. Cloning of the full-length SmGPCR
Total RNA was isolated from adult S. mansoni, using
TRIZOL™ reagent (Life Technologies Inc.), and passed
through an oligo (dT)-cellulose column (Amersham-
Pharmacia Biotech) to obtain purified poly (A+) mRNA.
An aliquot (0.5–1 ?g) of adult S. mansoni mRNA was
reverse-transcribed with an oligo-dT12–18primer and 200
U of murine Moloney leukemia virus reverse transcrip-
tase (RT) (Life Technologies, Inc). One tenth of the
resulting cDNA was used in a polymerase chain reaction
(PCR) (30 cycles, 30 s at 94 °C, 30 s at 48 °C, and 70
sat72 °C)containing20mMTris–HCl(pH8.4),50mM
KCl, 1.5 mM MgCl2, 0.2 mM dNTPs mix, 2 ?M of each
degenerate primer, and 2.5 U of Taq DNA polymerase
(Life Technologies, Inc). The two degenerate PCR
primers used in this amplification reaction were designed
to target consensus sequences of transmembrane two
(TM2) (VA/SVLVMP; sense: 5?-GTTKCIGTIYTKGT-
NATGCC-3?) and TM7 (WLG-YFNS; antisense: 5?-
GARTTRAARTANCCNARCCA-3?)
conserved among most biogenic amine GPCRs. An
aliquot (1/10) of the PCR product was subjected to
another 30 cycles of PCR as above, using the same
forward primer and a nested degenerate reverse primer
targeting conserved sequences in TM6 (CWL/FPFF;
5?-RAARAANGGRARCCARGA-3?). The PCR prod-
ucts were ligated to a T/A cloning vector (pCR2.1;
Invitrogen) and a total of 65 different clones were
hybridized, under low stringency, with a
DNA probe (Random Priming; Roche Molecular Bio-
chemicals) corresponding to the TM regions of the rat
5-HT1Areceptor [14], as described previously [15]. Four
positivecloneswereobtainedandDNAsequencedbythe
dideoxy chain termination method. One clone displayed
amino acid homology with various biogenic amine
GPCRs and was selected for further analysis.
To isolate the missing 5?-end of SmGPCR, we used an
anchored RT-PCR approach that targeted the conserved
5?-end spliced leader (SL) sequence of S. mansoni tran-
scripts [16,17], as described previously [15,18]. Briefly,
oligo dT reverse-transcribed cDNA was subjected
to a first nested PCR reaction with a SmGPCR-
specific antisense primer (primers A; see Fig. 1) and a
sense primer (primer SmSL; see Fig. 1), that corre-
sponded to nucleotides 9–32 of the S. mansoni 36
nucleotide SL sequence [16,17]. An aliquot (1/10) of the
thatarewell
32P-labeled

Page 3

F.F. Hamdan et al. / Molecular & Biochemical Parasitology 119 (2002) 75–8677
resulting product was then subjected to a second round
of PCR using the same SmSL primer and an internal
SmGPCR-specific antisense primer (primer B; Fig. 1).
The reaction conditions for PCR were as described
above and the cycling protocol for each reaction was 30
cycles of 30 s at 94 °C, 30 s at 53 °C and 90 s at 72 °C.
The final PCR product was cloned into pCR2.1 and
confirmed by DNA sequencing of two separate clones.
The remaining 3?-end of SmGPCR was obtained by a
standard rapid amplification of cDNA ends (RACE)
using a 3?-RACE kit (Life Technology, Inc), according
to the manufacturer’s recommendations. The RT was
performed as described above, except that an oligo(dT)-
adapter primer (supplied by the 3?-RACE kit) was used
to prime the reaction. An aliquot of the resulting
cDNA (0.2 ?l) was used in a standard PCR reaction (25
Fig. 1. SmGPCR complete cDNA and protein sequences. Transmembrane domains (boxed) were predicted according to the average results of
three different algorithms [38–40]. Codons that are of low usage in mammals are underlined. PCR primers (SmSL, A, B, C, D, S, and E) used
for cloning and RACE procedures are marked by a solid arrow. The S. mansoni SL sequence is italicized. Consensus N-glycosylation (N) and
phosphorylation sites (S, T) are encircled. The putative polyadenylation site is underlined and the stop codon is marked by an asterisk.

Page 4

F.F. Hamdan et al. / Molecular & Biochemical Parasitology 119 (2002) 75–8678
cycles, 30 s at 94 °C, 30 s at 53 °C, and 90 s at
72 °C) with a SmGPCR gene-specific sense primer
(primer C; see Fig. 1) and an antisense adapter
primer (supplied with the 3?-RACE kit). One tenth of
the resulting PCR product was subjected to a second
round of PCR using a nested gene-specific sense
primer (primer D; Fig. 1) and the same antisense
adapter primer. The final 3?-RACE product was
cloned into pCR2.1 and sequenced.
2.2. Construction of SmGPCR expression plasmids
The full-length SmGPCR was amplified directly
from oligodT reverse-transcribed S. mansoni cDNA
by a standard RT-PCR reaction, using primers that
targeted the beginning and the end of the coding se-
quence (primers S and E; see Fig. 1) and a DNA
proofreading polymerase (PWO; Roche Molecular
Biochemicals). The resulting product was subsequently
codon-optimized,according
codon usage, for expression in transfected HEK293
and COS7 cells. Briefly, the first 855 bp of the SmG-
PCR coding sequence was synthesized in a single re-
cursive PCR, as described [19], using 16 overlapping
mutagenic primers (eight sense primers and eight anti-
sense primers) designed to rewrite the S. mansoni
cDNA according to human preferred codons [20].
The resulting PCR product was ligated to the second
half of the SmGPCR coding sequence to produce a
continuous chimeric cDNA consisting of 855 bp of
codon-optimized sequence at the 5? end followed by
828 bp of wild-type SmGPCR sequence at the 3? end
for a complete cDNA. The mutagenesis was carefully
designed so as to ensure the final SmGPCR protein
product was unchanged by the codon modification.
Preliminary experiments indicated that rewriting the
first half of the SmGPCR cDNA was sufficient to
increase expression of the full-length receptor in
mammalian cells (data not shown). The ‘humanized’
SmGPCR cDNA was further modified by PCR, using
primers designed to introduce a FLAG epitope [21]
immediately preceded by a Kozak sequence (GC-
CACC) at the 5? end and a hexaoligohistidine tag,
followed by a stop codon and a single adenosine [22]
at the 3? end. The Kozak was required for optimal
translation initiation, whereas the single adenosine im-
mediately after the stop codon was designed to in-
crease translationtermination
malian cells [22]. The final product was ligated be-
tween the NheI/NotI sites of pCEP4 mammalian
expression vector (Invitrogen) or between the EcoRI
and
SmaI
sitesofexpression
(Promega). The two expression constructs, pCEP4-
SmGPCR and pCIneo–SmGPCR were confirmed by
DNA sequencing of at least two separate clones.
tohuman preferred
efficiencyin mam-
vector pCIneo
2.3. Indirect immunofluorescence
HEK293(EBNA1) cells (Invitrogen) were seeded
into six-well plates (2–3×105cells per well) contain-
ing sterilized coverslips, which were pre-coated with
0.001% poly-L-lysine (Sigma). Approximately 18–20 h
later, the cells were transfected with 1 ?g of plasmid
DNA (pCEP4–SmGPCR or pCEP4 alone) using Fu-
GENE 6 (Roche Molecular Biochemicals) (3 ?l), ac-
cordingto themanufacturer’s
About 48 h post-transfection, the cells were fixed with
4% paraformaldehyde (PFA) (Sigma) and incubated
(75 min at 4 °C) with a monoclonal antibody di-
rected against the FLAG epitope (anti-FLAGM2,
Sigma; 5 ?g ml−1), followed by a second incubation
(1 h at 4 °C) with a fluorescein isothiocyanate
(FITC)-conjugated antibody (goat anti-mouse IgG;
Sigma; 1:200 dilution). For experiments using a C-ter-
minal anti-His antibody (Invitrogen), cells were fixed
with PFA as above and used either directly, or after
permeabilization with ice-cold methanol for 7 min at
−20 °C. Cells were reacted with anti-His antibody
(1:200 dilution) for 1 h at 37 °C, prior to incubation
(1 h, 37 °C) with the FITC-conjugated secondary an-
tibody (1:200 dilution). Subsequently, the cells were
washed in phosphate buffer saline (PBS) and the cov-
erslips were mounted (PBS/glycerol, 1:1 v/v) on glass
slides and examined using fluorescence microscopy.
Indirect immunofluorescence analyses of SmGPCR
expressed in COS7 cells were performed in the same
way except that cells were transfected with 1 ?g of a
pCIneo expression construct (pCIneo–SmGPCR) or
pCIneo alone for mock-transfected controls.
recommendations.
2.4. Secreted alkaline phosphatase (CRE-SEAP)
reporter assays
Cells (293CRE-SEAP) [23] were seeded in 96-well
plates (approximately 104cells per well) and were
transiently transfectedwith
pCEP4–SmGPCR or pCEP4 vector alone (50 ng per
well), using FuGENE 6 reagent, according to stan-
dard procedures. 293CRE-SEAP cells were derived
from HEK293(EBNA1) cells that were stably trans-
fected with a secreted form of alkaline phosphatase
(SEAP) under the control of a cAMP-responsive ele-
ment (CRE) [23]. Twenty four hours post-transfec-
tion, cells were treated with test agents for 6 or 16 h
at 37 °C in the presence and absence of 2, 5 or 10
?M forskolin or 100 ?M IBMX, for Gi/o or Gs read-
outs, respectively. Aliquots (50 ?l) of the medium
containing SEAP were assayed directly for enzyme
activity by using a colorimetric method and a plate
format absorbance reader set at 405 nm, as described
previously [23].
expression plasmid,

Page 5

F.F. Hamdan et al. / Molecular & Biochemical Parasitology 119 (2002) 75–8679
2.5. Aqueorin assays
COS7 cells were transiently cotransfected with an
aqueorin expression vector (pCDM.AEQ; licensed from
the NIH, Bethesda, MD) [24], and either a SmGPCR
expression vector (pCIneo–SmGPCR or pCEP–SmG-
PCR) or vector that lacked insert. Transfected cells
were charged with coelenterazine (8 ?M) approximately
48 h post-transfection and then dispensed into 96-well
plates (5×104cells per well) containing different con-
centrations of test drugs. Light emission was recorded
with a Luminoskan RS luminometer (Labsystems, Nee-
ham Heights, MA) as described previously [25,26].
2.6. Mutagenesis of SmGPCR Asn111
Expression construct pCIneo–SmGPCR containing
the codon optimized, double tagged SmGPCR cDNA,
was used in a site-directed mutagenesis reaction using a
mutagenic primer designed to change Asn111(AAT) to
an aspartate residue (GAT). The mutagenesis was done
with the use of a commercial kit (Quick Change, Strata-
gene), according to the recommendations of the manu-
facturer. The mutant (pCIneo–SmGPCR.Asp111) was
confirmed by DNA sequencing and tested for activity
by using an aqueorin assay in transiently transfected
COS7 cells, as described above.
3. Results
3.1. Cloning of SmGPCR
A partial cDNA sequence was first amplified by
degenerate RT-PCR and found to have sequence ho-
mology with various biogenic monoamine receptors.
The missing 3? end was subsequently obtained by a
standard 3? RACE procedure [27] and the 5? end was
amplified in an anchored RT-PCR reaction that
targeted the conserved SL sequence of S. mansoni
[16,17]. Fig. 1 shows the complete nucleotide sequence
and predicted amino acid sequence of SmGPCR. The
2002 bp cDNA includes 267 bp of 5?-flanking region
starting with the SL sequence, followed by a single
open reading frame of 1683 bp (nucleotides 268–1950)
and a short 3? untranslated region (3?-UTR) of about 52
bp. It is noteworthy that SmGPCR exhibits a complete
S. mansoni splice leader, including the last four nucle-
otides (CATG; Fig. 1), which were not part of the
SmSL primer used in the anchored PCR reaction. This
confirms that SmGPCR is trans-spliced at the 5?-end to
the S. mansoni SL, just as previously described for
several other cDNAs. At the 3? end, SmGPCR carries a
consensus polyadenylation site (AATAAA) upstream
of a poly(A) tail, and thus is presumed to be full length.
3.2. Protein sequence analysis of SmGPCR
SmGPCR encodes a predicted protein of 560 amino
acids with a calculated molecular mass of approxi-
mately 65 kDa. Hydropathy analyses of the deduced
amino acid sequence identified seven potential TM
domains (Fig. 1) connected by predicted intra- and
extracellular loops of variable lengths, an organization
which is characteristic of all class I GPCRs. In addi-
tion, SmGPCR exhibits several distinctive GPCR mo-
tifs, for example the nearly invariant proline residues of
TM5 and TM6 (SmGPCR residues Pro200, and Pro494,
respectively), an aspartate in TM2 (Asp76), the ‘DRY’
motif at the C-terminal end of TM3 (positions 128–
130) and the highly conserved ‘NPX2Y’ motif of TM7
(positions 528–532) reviewed in [28]. Two conserved
cysteines in the first and the second extracellular loops
(Cys104and Cys180) are predicted to be linked by a
disulfide bond [28]. In addition, SmGPCR has two
consensus sites for N-linked glycosylation (Asn5and
Asn10) at the N-terminal end and several putative phos-
phorylation sites for protein kinase C (Thr338, Ser542)
and calcium-calmodulin dependent protein kinase type
2 (Ser343and Ser443) in the predicted intracellular loops
and C-terminal end (Fig. 1).
3.3. SmGPCR is an ‘orphan’ receptor
A Position-Specific Iterated BLAST (PSI-BLAST)
analysis at the amino acid level [29] showed that SmG-
PCR is related primarily to biogenic amine receptors
and muscarininc acetylcholine receptors. Additional
CLUSTAL [30] pairwise comparisons revealed about
the same level of moderate sequence homology (?
35%) with histamine receptors, octopamine/tyramine
receptors and adrenergic receptors, and slightly lower
homology (25–30%) with other types of monoamine
GPCRs, including serotonergic, dopamine and mus-
carinic acetylcholine receptors. The level of homology
with the various sequences increased to an average of
51–55%, when the analysis was restricted to predicted
TM domains, which are typically more conserved
among all class I GPCRs. A dendogram analysis of
nearly 50 monoamine GPCR sequences (complete se-
quences), including representatives from major receptor
classes, showed that the S. mansoni receptor was dis-
tantly related to histamine GPCRs, particularly H1,
H3, and H4, but did not cluster exclusively with any
one group of receptors (Fig. 2). The overall level of
sequence homology with H1, H3, H4 and H2 was 35,
30, 30 and 25%, respectively. An alignment of SmG-
PCR with the four mammalian histamine classes iden-
tified important substitutions at positions previously
implicated in histamine receptor binding and activation,
most notably Asn111, which replaces a highly conserved
aspartate of TM3, and Gly196in place of a charged